Salts and processes of preparing a pi3k inhibitor

ABSTRACT

The present application provides processes for preparing (R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo [3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one, which is useful as an inhibitor phosphoinositide 3-kinase-delta (PI3Kδ), as well as a salt form and intermediates related thereto.

This application is a divisional of U.S. Ser. No. 15/054,474, filed Feb.26, 2016, which claims the benefit of U.S. Ser. No. 62/121,697, filedFeb. 27, 2015, both of which are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

The present application provides process for preparing(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one,which is useful as an inhibitor of phosphoinositide 3-kinase-delta(PI3Kδ), as well as a salt form and intermediates related thereto.

BACKGROUND

The phosphoinositide 3-kinases (PI3Ks) belong to a large family of lipidsignaling kinases that phosphorylate phosphoinositides at the D3position of the inositol ring (Cantley, Science, 2002, 296(5573):1655-7). PI3Ks are divided into three classes (class I, II, and III)according to their structure, regulation and substrate specificity.Class I PI3Ks, which include PI3α, PI3Kβ, PI3Kγ, and PI3Kδ, are a familyof dual specificity lipid and protein kinases that catalyze thephosphorylation of phosphatidylinosito-4,5-bisphosphate (PIP₂) givingrise to phosphatidylinosito-3,4,5-trisphosphate (PIP₃). PIP₃ functionsas a second messenger that controls a number of cellular processes,including growth, survival, adhesion and migration. All four class IPI3K isoforms exist as heterodimers composed of a catalytic subunit(p110) and a tightly associated regulatory subunit that controls theirexpression, activation, and subcellular localization. PI3Kα, PI3Kβ, andPI3Kδ associate with a regulatory subunit known as p85 and are activatedby growth factors and cytokines through a tyrosine kinase-dependentmechanism (Jimenez, et al., J Biol Chem., 2002, 277(44):41556-62)whereas PI3Kγ associates with two regulatory subunits (p101 and p84) andits activation is driven by the activation of G-protein-coupledreceptors (Brock, et al., J Cell Biol., 2003, 160(1):89-99). PI3Kα andPI3Kβ are ubiquitously expressed. In contrast, PI3Kγ and PI3Kδ arepredominantly expressed in leukocytes (Vanhaesebroeck, et al., TrendsBiochem Sci., 2005, 30(4):194-204).

The differential tissue distribution of the PI3K isoforms factors intheir distinct biological functions. Genetic ablation of either PI3Kα orPI3Kβ results in embryonic lethality, indicating that PI3Kα and PI3Kβhave essential and non-redundant functions, at least during development(Vanhaesebroeck, et al., 2005). In contrast, mice which lack PI3Kγ andPI3Kδ are viable, fertile and have a normal life span although they showan altered immune system. PI3Kγ deficiency leads to impaired recruitmentof macrophages and neutrophils to sites of inflammation as well asimpaired T cell activation (Sasaki, et al., Science, 2000,287(5455):1040-6). PI3Kδ-mutant mice have specific defects in B cellsignaling that lead to impaired B cell development and reduced antibodyresponses after antigen stimulation (Clayton, et al., J Exp Med. 2002,196(6):753-63; Jou, et al., Mol Cell Biol. 2002, 22(24):8580-91;Okkenhaug, et al., Science, 2002, 297(5583):1031-4).

The phenotypes of the PI3Kγ and PI3Kδ-mutant mice suggest that theseenzymes may play a role in inflammation and other immune-based diseasesand this is borne out in preclinical models. PI3Kγ-mutant mice arelargely protected from disease in mouse models of rheumatoid arthritis(RA) and asthma (Camps, et al., Nat Med. 2005, 11(9):936-43; Thomas, etal., Eur. J. Immunol. 2005, 35(4):1283-91). In addition, treatment ofwild-type mice with a selective inhibitor of PI3Kγ was shown to reduceglomerulonephritis and prolong survival in the MRL-lpr model of systemiclupus nephritis (SLE) and to suppress joint inflammation and damage inmodels of RA (Barber, et al., Nat Med. 2005, 11(9):933-5; Camps, et al.,2005). Similarly, both PI3Kδ-mutant mice and wild-type mice treated witha selective inhibitor of PI3Kδ have been shown to have attenuatedallergic airway inflammation and hyper-responsiveness in a mouse modelof asthma (Ali, et al., Nature. 2004, 431(7011):1007-11; Lee, et al.,FASEB J. 2006, 20(3):455-65) and to have attenuated disease in a modelof RA (Randis, et al., Eur. J. Immunol., 2008, 38(5):1215-24).

B cell proliferation has shown to play a major role in the developmentof inflammatory autoimmune diseases (Puri, Frontiers in Immunology(2012), 3(256), 1-16; Walsh, Kidney International (2007) 72, 676-682).For example, B cells support T-cell autoreactivity, an importantcomponent of inflammatory autoimmune diseases. Once activated andmatured, B cells can traffic to sites of inflammation and recruitinflammatory cells or differentiate to plasmablasts. Thus, activity ofB-cells can be affected by targeting B-cell stimulatory cytokines,B-cell surface receptors, or via B-cell depletion. Rituximab—an IgG1 κmouse/human chimeric monoclonal antibody directed against the B-cellsurface receptor CD20—has been shown to deplete CD20+ B cells. Use ofrituximab has been shown to have efficacy in treating idiopathicthrombocytopenic purpura, autoimmune hemolytic anemia, or vasculitis.For example, treatment with rituximab resulted in remission of thedisease in patients suffering from anti-neutrophil cytoplasm antibodyassociated (ANCA) systemic vasculitis (AASV) with demonstratedperipheral B-cell depletion (Walsh, 2007; Lovric, Nephrol DialTransplant (2009) 24: 179-185). Similarly, a complete response wasreported in one-third to two-thirds of patients having mixedcryoglobulinemia vasculitis after treatment with rituximab, includingpatients who presented with a severe form of vasculitis that wasresistant or intolerant to other treatments (Cacoub, Ann Rheum Dis 2008;67:283-287). Similarly, rituximab has been shown to have efficacy intreating patients with idiopathic thrombocytopenic purpura (or immunethrombocytopenic purpura) (Garvey, British Journal of Haematology,(2008) 141, 149-169; Godeau, Blood (2008), 112(4), 999-1004; Medeo,European Journal of Haematology, (2008) 81, 165-169) and autoimmunehemolytic anemia (Garvey, British Journal of Haematology, (2008) 141,149-169).

PI3Kδ signaling has been tied to B cell survival, migration, andactivation (Puri, Frontiers in Immunology, 2012, 3(256), 1-16, at pages1-5; and Clayton, J Exp Med, 2002, 196(6):753-63). For example, PI3Kδ isrequired for antigen-dependent B-cell activation driven by B cellreceptor. By blocking B-cell adhesion, survival, activation, andproliferation, PI3Kδ inhibition can impair the ability of B cells toactivate T cells, preventing their activation and reducing secretion ofautoantibodies and pro-inflammatory cytokines. Hence, by their abilityto inhibit B cell activation, PI3Kδ inhibitors would be expected totreat B cell mediated diseases that were treatable by similar methodssuch as B cell depletion by rituximab. Indeed, PI3Kδ inhibitors havebeen shown to be useful mouse models of various autoimmune diseases thatare also treatable by rituximab such as arthritis (Puri (2012)).Further, innate-like B cells, which are linked to autoimmunity aresensitive to PI3Kδ activity, as MZ and B-1 cells are nearly absent inmice lacking the p110δ gene (Puri (2012). PI3Kδ inhibitors can reducetrafficking of and activation of MZ and B-1 cells, which are implicatedin autoimmune diseases.

In addition to their potential role in inflammatory diseases, all fourclass I PI3K isoforms may play a role in cancer. The gene encoding p110αis mutated frequently in common cancers, including breast, prostate,colon and endometrial (Samuels, et al., Science, 2004, 304(5670):554;Samuels, et al., Curr Opin Oncol. 2006, 18(1):77-82). Eighty percent ofthese mutations are represented by one of three amino acid substitutionsin the helical or kinase domains of the enzyme and lead to a significantupregulation of kinase activity resulting in oncogenic transformation incell culture and in animal models (Kang, et al., Proc Natl Acad Sci USA.2005, 102(3):802-7; Bader, et al., Proc Natl Acad Sci USA. 2006,103(5):1475-9). No such mutations have been identified in the other PI3Kisoforms although there is evidence that they can contribute to thedevelopment and progression of malignancies. Consistent overexpressionof PI3Kδ is observed in acute myeloblastic leukemia (Sujobert, et al.,Blood, 2005, 106(3): 1063-6) and inhibitors of PI3Kδ can prevent thegrowth of leukemic cells (Billottet, et al., Oncogene. 2006,25(50):6648-59). Elevated expression of PI3Kγ is seen in chronic myeloidleukemia (Hickey, et al., J Biol. Chem. 2006, 281(5):2441-50).Alterations in expression of PI3Kβ, PI3Kγ and PI3Kδ have also beenobserved in cancers of the brain, colon and bladder (Benistant, et al.,Oncogene, 2000, 19(44):5083-90; Mizoguchi, et al., Brain Pathol. 2004,14(4):372-7; Knobbe, et al., Neuropathol Appl Neurobiol. 2005,31(5):486-90). Further, these isoforms have all been shown to beoncogenic in cell culture (Kang, et al., 2006).

For these reasons, there is a need to develop new PI3K inhibitors thatcan be used inflammatory disorders, autoimmune diseases and cancer. Thisinvention is directed to this need and others.

SUMMARY

The present application provides processes of preparing a compound ofFormula I:

or a pharmaceutically acceptable salt thereof, which is useful as aninhibitor of PI3Kδ.

The present application further provides a hydrochloric acid salt of thecompound of Formula I.

The present application also provides pharmaceutical compositionscomprising a hydrochloric acid salt described herein and apharmaceutically acceptable carrier.

The present application further provides methods of inhibiting anactivity of a PI3K kinase, comprising contacting the kinase with thehydrochloric acid salt of the compound of Formula I.

The present application also provides methods of treating a disease in apatient, wherein said disease is associated with abnormal expression oractivity of a PI3K kinase, comprising administering to said patient atherapeutically effective amount of the hydrochloric acid salt of thecompound of Formula I.

The present application additionally provides the hydrochloric acid saltof the compound of Formula I for use in any of the methods describedherein.

The present application further provides use of the hydrochloric acidsalt of the compound of Formula I for the manufacture of a medicamentfor use in any of the methods described herein.

The present application also provides a process of preparing thehydrochloric acid salt of the compound of Formula I, comprising reactinga compound of Formula I:

with hydrochloric acid to form said salt.

The present application additionally provides a process of preparing acompound of Formula I, comprising reacting a compound of Formula XVI:

with formamidine acetate to form said compound of Formula I:

The present application further provides a compound of Formula XIV:

or a pharmaceutically acceptable salt thereof.

The present application also provides a compound of Formula XV:

or a pharmaceutically acceptable salt thereof.

The present application additionally provides a compound of Formula XVI:

or a pharmaceutically acceptable salt thereof.

The present application further provides a compound of Formula XIX:

or a pharmaceutically acceptable salt thereof.

The present application also provides a compound of Formula XX:

or a pharmaceutically acceptable salt thereof.

The present application additionally provides a compound of Formula XXI:

or a pharmaceutically acceptable salt thereof.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a DSC thermogram representative of the salt of Example 3.

FIG. 2 shows TGA data representative of the salt of Example 3.

FIG. 3 shows an XRPD pattern representative of the salt of Example 3.

DETAILED DESCRIPTION Compounds and Salts

The present application provides processes of preparing a compound ofFormula I:

or a pharmaceutically acceptable salt thereof, which is useful as aninhibitor of PI3Kδ, wherein Et is ethyl.

The present application further provides a salt of the compound ofFormula I.

Accordingly, in some embodiments, the present application provides4-(3-(1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloric acid salt. In some embodiments, the present applicationprovides(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloric acid salt. In some embodiments, the salt is a 1:1stoichiometric ratio of(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-oneto hydrochloric acid.

Different forms of the same substance have different bulk propertiesrelating to, for example, hygroscopicity, solubility, stability, and thelike. Forms with high melting points often have good thermodynamicstability which is advantageous in prolonging shelf-life drugformulations containing the solid form. Forms with lower melting pointsoften are less thermodynamically stable, but are advantageous in thatthey have increased water solubility, translating to increased drugbioavailability. Forms that are weakly hygroscopic are desirable fortheir stability to heat and humidity and are resistant to degradationduring long storage.

In some embodiments, the hydrochloric acid salt of the compound ofFormula I provided herein is crystalline. As used herein, “crystalline”or “crystalline form” is meant to refer to a certain latticeconfiguration of a crystalline substance. Different crystalline forms ofthe same substance typically have different crystalline lattices (e.g.,unit cells) which are attributed to different physical properties thatare characteristic of each of the crystalline forms. In some instances,different lattice configurations have different water or solventcontent.

The different salt forms can be identified by solid statecharacterization methods such as by X-ray powder diffraction (XRPD).Other characterization methods such as differential scanning calorimetry(DSC), thermogravimetric analysis (TGA), dynamic vapor sorption (DVS),and the like further help identify the form as well as help determinestability and solvent/water content.

An XRPD pattern of reflections (peaks) is typically considered afingerprint of a particular crystalline form. It is well known that therelative intensities of the XRPD peaks can widely vary depending on,inter alia, the sample preparation technique, crystal size distribution,various filters used, the sample mounting procedure, and the particularinstrument employed. In some instances, new peaks may be observed orexisting peaks may disappear, depending on the type of the instrument orthe settings. As used herein, the term “peak” refers to a reflectionhaving a relative height/intensity of at least about 4% of the maximumpeak height/intensity. Moreover, instrument variation and other factorscan affect the 2-theta values. Thus, peak assignments, such as thosereported herein, can vary by plus or minus about 0.2° (2-theta), and theterm “substantially” and “about” as used in the context of XRPD hereinis meant to encompass the above-mentioned variations.

In some embodiments, the hydrochloric acid salt of the compound ofFormula I has at least one XRPD peak, in terms of 2-theta, selected fromabout 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28.10, about31.2°, and about 32.8°. In some embodiments, the hydrochloric acid saltof the compound of Formula I has at least two XRPD peaks, in terms of2-theta, selected from about 11.3°, about 16.4°, about 21.0°, about23.0°, about 28.10, about 31.2°, and about 32.8°. In some embodiments,the hydrochloric acid salt of the compound of Formula I has at leastthree XRPD peaks, in terms of 2-theta, selected from about 11.3°, about16.4°, about 21.0°, about 23.0°, about 28.1°, about 31.2°, and about32.8°. In some embodiments, the hydrochloric acid salt of the compoundof Formula I has at least four XRPD peaks, in terms of 2-theta, selectedfrom about 11.3°, about 16.4°, about 21.0°, about 23.0°, about 28.10,about 31.2°, and about 32.8°. In some embodiments, the hydrochloric acidsalt of the compound of Formula I has at least five XRPD peaks, in termsof 2-theta, selected from about 11.3°, about 16.4°, about 21.0°, about23.0°, about 28.10, about 31.2°, and about 32.8°. In some embodiments,the hydrochloric acid salt of the compound of Formula I has an XRPDprofile substantially as shown in FIG. 3.

In the same way, temperature readings in connection with DSC, TGA, orother thermal experiments can vary about +3° C. depending on theinstrument, particular settings, sample preparation, etc. Accordingly, acrystalline form reported herein having a DSC thermogram “substantially”as shown in any of the Figures or the term “about” is understood toaccommodate such variation. In some embodiments, the hydrochloric acidsalt of the compound of Formula I has a DSC thermogram having anendothermic peak at about 207° C. In some embodiments, the hydrochloricacid salt of the compound of Formula I has a DSC thermogramsubstantially as shown in FIG. 1. In some embodiments, the hydrochloricacid salt of the compound of Formula I has a TGA thermogramsubstantially as shown in FIG. 2.

In some embodiments, the salts and compounds described herein (e.g., thecompound of Formula I or the hydrochloric acid salt of the compound ofFormula I) are substantially isolated. By “substantially isolated” ismeant that the salt or compound is at least partially or substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe salts described herein. Substantial separation can includecompositions containing at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 97%, or at least about 99% by weight of the saltsdescribed herein, or salt thereof. Methods for isolating compounds andtheir salts are routine in the art.

Intermediates

The present application further provides intermediates that are usefulin the preparation of the compound of Formula I.

Accordingly, in some embodiments, the present application provides acompound of Formula XIV:

or a pharmaceutically acceptable salt thereof.

The present application further provides a compound of Formula XV:

or a pharmaceutically acceptable salt thereof.

The present application further provides a compound of Formula XVI:

or a pharmaceutically acceptable salt thereof.

The present application further provides a compound of Formula XIX:

or a pharmaceutically acceptable salt thereof.

The present application further provides a compound of Formula XX:

or a pharmaceutically acceptable salt thereof.

The present application further provides a compound of Formula XXI:

or a pharmaceutically acceptable salt thereof.

Processes

The present application further provides a process of preparing a saltof Formula I:

In some embodiments, the process comprises reacting a compound ofFormula I:

with hydrochloric acid to form said salt.

In some embodiments, said hydrochloric acid is 1 M aqueous hydrochloricacid.

In some embodiments, about 3.3 to about 3.7 equivalents of hydrochloricacid is used based on 1 equivalent of the compound of Formula I.

In some embodiments, said reacting is performed at a temperature of fromabout 45° C. to about 55° C.

In some embodiments, the process comprises:

adding hydrochloric acid to the compound of Formula I at roomtemperature to form a slurry;

heating said slurry to a temperature of from about 45° C. to about 55°C. to form a solution; and

cooling the solution to a temperature of from about 0° C. to about 5° C.to crystallize said salt.

In some embodiments, the process comprises reacting a compound ofFormula XVI:

with formamidine acetate to form a compound of Formula I:

In some embodiments, said reacting of the compound of Formula XVI withformamidine acetate is conducted in a solvent component comprising1,2-ethanediol.

In some embodiments, said reacting of the compound of Formula XVI withformamidine acetate is performed at a temperature of from about 100° C.to about 105° C.

In some embodiments, about 8 to about 10 equivalents of formamidineacetate is used based on 1 equivalent of the compound of Formula XVI.

In some embodiments, the process further comprises preparing thecompound of Formula XVI by a process comprising reacting a compound ofFormula XV:

with (1-ethoxyethylidene)malononitrile in the presence of a tertiaryamine.

In some embodiments, said tertiary amine is N-methylpyrrolidinone.

In some embodiments, said reacting of the compound of Formula XV withwith (1-ethoxyethylidene)malononitrile is performed at about roomtemperature.

In some embodiments, the process further comprises preparing thecompound of Formula XV by a process comprising reacting a compound ofFormula XIV-a:

with hydrazine in the presence of a tertiary amine, wherein P¹ is C₁₋₆alkylsulfonyl.

In some embodiments, said tertiary amine is N-methylpyrrolidinone.

In some embodiments, said reacting of the compound of Formula XIV-a withhydrazine is performed at a temperature of from about 35° C. to about60° C.

In some embodiments, said reacting of the compound of Formula XIV-a withhydrazine is conducted in a solvent component comprisingdichloromethane.

In some embodiments, P¹ is methanesulfonyl group.

In some embodiments, the process further comprises preparing thecompound of Formula XIV by a process comprising reacting a compound ofFormula XIII:

with C₁₋₆ alkylsulfonylhalide in the presence of a tertiary amine.

In some embodiments, said C₁₋₆ alkylsulfonylhalide is methanesulfonylchloride.

In some embodiments, said tertiary amine is N,N-diisopropylethylamine.

In some embodiments, about 1.1 to about 1.5 equivalents ofalkylsulfonylhalide is used based on 1 equivalent of the compound ofFormula XIII.

In some embodiments, said reacting of said compound of Formula XIII withC₁₋₆ alkylsulfonylhalide is performed at a temperature of from about−10° C. to about 5° C.

In some embodiments, said reacting of said compound of Formula XIII withC₁₋₆ alkylsulfonylhalide is performed in a solvent component comprisingdichloromethane.

In some embodiments, the steps of: (i) reacting of said compound ofFormula XIII with C₁₋₆ alkylsulfonylhalide; (ii) reacting said compoundof Formula XIV-a with hydrazine in the presence of a tertiary amine toform a compound of Formula XV; and (iii) reacting said compound ofFormula XV with formamidine acetate to form a compound of Formula XVIare conducted in the same pot without isolation of the compound ofFormula XIV-a or the compound of Formula XV.

In some embodiments, the process further comprises preparing thecompound of Formula XVI by a process comprising reacting a salt ofFormula XV-a:

with (1-ethoxyethylidene)malononitrile in the presence of a tertiaryamine, wherein TsOH is p-toluenesulfonic acid.

In some embodiments, said tertiary amine is N,N-diisopropylethylamine.

In some embodiments, said reacting a salt of Formula XV-a with with(1-ethoxyethylidene)malononitrile is performed at about roomtemperature.

In some embodiments, about 1.3 to about 1.6 equivalents of(1-ethoxyethylidene)malononitrile is used based on 1 equivalent of thesalt of Formula XV-a.

In some embodiments, said reacting of the salt of Formula XV-a with with(1-ethoxyethylidene)malononitrile is conducted in a solvent componentcomprising ethanol.

In some embodiments, the process further comprising preparing the saltof Formula XV-a by a process comprising reacting a compound of FormulaXXI:

with p-toluenesulfonic acid, wherein Boc is tert-butoxycarbonyl.

In some embodiments, said p-toluenesulfonic acid is p-toluenesulfonicacid monohydrate.

In some embodiments, about 1.3 to about 1.6 equivalents ofp-toluenesulfonic acid is used based on 1 equivalent of the compound ofFormula XXI.

In some embodiments, said reacting of said compound of Formula XXI withp-toluenesulfonic acid is performed at a temperature of from about 45°C. to about 65° C.

In some embodiments, reacting of said compound of Formula XXI withp-toluenesulfonic acid is conducted in a solvent component comprisingethanol.

In some embodiments, the steps of: (i) reacting said compound of FormulaXXI with p-toluenesulfonic acid to form a salt of Formula XV-a; and (ii)reacting said salt of Formula XV-a with(1-ethoxyethylidene)malononitrile are conducted in the same pot withoutisolation of the salt of Formula XV-a.

In some embodiments, the process further comprises preparing thecompound of Formula XXI by a process comprising reacting a compound ofFormula XX:

with hydrogen gas in the presence of one or more independently selectedhydrogenation catalysts, wherein Boc is tert-butoxycarbonyl.

In some embodiments, said reacting of the compound of Formula XX withhydrogen gas is performed in the presence of two independently selectedhydrogenation catalysts.

In some embodiments, one hydrogenation catalyst isbis(1,5-cyclooctadiene)rhodium(I)tetrafluoroborate and the other is(R)-(−)-1-{(S)-2-[bis(4-trifluoromethylphenyl)phosphine]ferrocenyl}ethyl-di-t-butylphosphine.

In some embodiments, about 13.5 to about 14.5 equivalents ofbis(1,5-cyclooctadiene)rhodium(I)tetrafluoroborate is used based on 1equivalent of the compound of Formula XX.

In some embodiments, about 12 to about 13 equivalents of(R)-(−)-1-{(S)-2-[bis(4-trifluoromethylphenyl)phosphine]ferrocenyl}ethyl-di-t-butylphosphineis used based on 1 equivalent of the compound of Formula XX.

In some embodiments, said reacting of the compound of Formula XX withhydrogen gas is performed at about room temperature.

In some embodiments, said reacting of the compound of Formula XX withhydrogen gas is conducted in a solvent component comprising methanol.

In some embodiments, the process further comprises preparing thecompound of Formula XX by a process comprising reacting a compound ofFormula XIX:

with t-butyl carbazate.

In some embodiments, said reacting of the compound of Formula XIX witht-butyl carbazate is performed at a temperature of from about 60° C. toabout 70° C.

In some embodiments, said reacting of the compound of Formula XIX witht-butyl carbazate is conducted in a solvent component comprisingmethanol.

In some embodiments, the process further comprises preparing thecompound of Formula XIX by a process comprising oxidizing a compound ofFormula XIII-a:

in the presence of an oxidizing agent.

In some embodiments, said oxidizing agent is Dess-Martin periodinane.

In some embodiments, about 1.2 to about 1.7 equivalents of saidoxidizing agent is used based on 1 equivalent of the compound of FormulaXIII-a.

In some embodiments, said oxidizing of the compound of Formula XIII-a isperformed at about room temperature.

In some embodiments, said oxidizing of the compound of Formula XIII-a isconducted in a solvent component comprising dichloromethane.

In some embodiments, said compound of Formula XIII is prepared by aprocess comprising heating a compound of Formula XII:

in the presence of a solvent component.

In some embodiments, said heating is performed at a temperature of fromabout 95° C. to about 105° C.

In some embodiments, said solvent component comprises 1,4-dioxane.

In some embodiments, said solvent component comprises toluene.

In some embodiments, said solvent component comprises 1,4-dioxane andtoluene.

In some embodiments, said compound of Formula XII is prepared by aprocess comprising reacting a compound of Formula XI:

in the presence of a strong base.

In some embodiments, said strong base is sodium hydroxide.

In some embodiments, said strong base is 1 M aqueous sodium hydroxide.

In some embodiments, said reacting is performed at about roomtemperature.

In some embodiments, said compound of Formula XI is prepared by aprocess comprising reacting a compound of Formula X:

with hydrogen gas in the presence of Raney nickel.

In some embodiments, said reacting is performed at a temperature of fromabout 50° C. to about 70° C.

In some embodiments, said compound of Formula X is prepared by a processcomprising reacting a compound of Formula IX:

in the presence of boron trifluoride etherate,(3aS)-1-methyl-3,3-diphenyltetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaborole((S)-MeCBS) catalyst, and a borane complex.

In some embodiments, about 0.03 to about 0.07 equivalents of borontrifluoride etherate is used based on 1 equivalent of the compound ofFormula IX.

In some embodiments, about 0.05 to about 0.15 equivalents of(3aS)-1-methyl-3,3-diphenyltetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaborole((S)-MeCBS) catalyst is used based on 1 equivalent of the compound ofFormula IX.

In some embodiments, said borane complex is 1.0 M borane-THF complex inTHF.

In some embodiments, about 0.9 to about 1.1 equivalents of boranecomplex is used based on 1 equivalent of the compound of Formula IX.

In some embodiments, said reacting is performed at about roomtemperature.

In some embodiments, said compound of Formula IX is prepared by aprocess comprising reacting a compound of Formula VIII:

with iodine in the presence of a solvent component.

In some embodiments, said solvent component comprises acetone.

In some embodiments, about 0.75 to about 1.25 equivalents of iodine isused based on 1 equivalent of the compound of Formula VIII.

In some embodiments, said compound of Formula VIII is prepared by aprocess comprising reacting a compound of Formula VII:

with dimethyl malonate in the presence of a catalyst.

In some embodiments, said catalyst is(1S,2S)—N,N′-dibenzylcyclohexane-1,2-diamine-dibromonickel (Evans'Catalyst).

In some embodiments, about 1.1 to about 1.3 equivalents of dimethylmalonate is used based on 1 equivalent of the compound of Formula VII.

In some embodiments, about 0.02 to about 0.03 equivalents of transitionmetal catalyst is used base on 1 equivalent of the compound of FormulaVII.

In some embodiments, said compound of Formula VII is prepared by aprocess comprising reacting a compound of Formula VI:

with nitromethane in the presence of an organic acid to form a firstmixture.

In some embodiments, said organic acid is glacial acetic acid.

In some embodiments, about 9.5 to about 10.5 equivalents of nitromethaneis used based on 1 equivalent of the compound of Formula VI.

In some embodiments, said reacting further comprises adding an aminebase to said first mixture to form a second mixture.

In some embodiments, said amine base is benzylamine.

In some embodiments, about 0.2 to about 0.3 equivalents of amine base isused based on 1 equivalent of the compound of Formula VI.

In some embodiments, said second mixture is heated at about 55° C. toabout 65° C.

In some embodiments, said compound of Formula VI is prepared by aprocess comprising reacting a compound of Formula V:

with a (C₁₋₄ alkyl)magnesiumhalide complex to form a first mixture.

In some embodiments, said (C₁₋₄ alkyl)magnesium halide complex is 1.3 Misopropylmagnesium chloride lithium chloride complex.

In some embodiments, about 1.1 to about 1.3 equivalents of said (C₁₋₄alkyl)magnesiumhalide complex is used based on 1 equivalent of thecompound of Formula V.

In some embodiments, said reacting further comprises addingN-formylmorpholine to said first mixture to form a second mixture.

In some embodiments, about 1.8 to about 2.2 equivalents ofN-formylmorpholine is used based on 1 equivalent of the compound ofFormula V.

In some embodiments, said reacting is performed at a temperature of fromabout −5° C. to about 10° C.

In some embodiments, said compound of Formula V is prepared according toprocedures described in U.S. Publication No. 2013-0059835A1.

In some embodiments, said compound of Formula VI is prepared by aprocess comprising reacting a compound of Formula V-a:

with N,N-dimethylformamide in the presence of lithium diisopropylamide.

In some embodiments, said lithium diisopropylamide is prepared byreacting N,N-diisopropylamine in the presence of n-butyllithium.

In some embodiments, said lithium diisopropylamide is prepared at atemperature of from about −75° C. to about 5° C.

In some embodiments:

(ii) said compound of Formula V-a is reacted with lithiumdiisopropylamide to form a first mixture; and

(ii) N,N-dimethylformamide is added to said first mixture to form asecond mixture.

In some embodiments, about 1.2 to about 1.3 equivalents of amine base isused based on 1 equivalent of the compound of Formula V-a.

In some embodiments, about 1.4 to about 1.6 equivalents ofN,N-dimethylformamide is used based on 1 equivalent of the compound ofFormula V-a.

In some embodiments, said compound of Formula V-a is prepared by aprocess comprising reacting a compound of Formula IV-a:

with 1,2-ethanediol in the presence of p-toluenesulfonic acid.

In some embodiments, said p-toluenesulfonic acid is p-toluenesulfonicacid monohydrate.

In some embodiments, about 2.2 to about 2.7 equivalents of1,2-ethanediol is used based on 1 equivalent of the compound of FormulaIV-a.

In some embodiments, about 0.05 to about 0.1 equivalents ofp-toluenesulfonic acid is used based on 1 equivalent of the compound ofFormula IV-a.

In some embodiments, said reacting is performed at about reflux.

In some embodiments, said compound of Formula IV-a is prepared by aprocess comprising reacting a compound of Formula II:

with CH₂CH₂—X¹ in the presence of an alkali metal carbonate base,wherein:

X¹ is halide.

In some embodiments, X¹ is iodide.

In some embodiments, said alkali metal carbonate base is potassiumcarbonate.

In some embodiments, about 1.1 to about 1.3 equivalents of CH₂CH₂—X¹ isused based on 1 equivalent of the compound of Formula II.

In some embodiments, about 1.8 to about 2.2 equivalents of alkali metalcarbonate base is used based on 1 equivalent of the compound of FormulaII.

In some embodiments, said reacting is performed at about 55° C. to about65° C.

In some embodiments, said compound of Formula II is prepared accordingto procedures described in U.S. Publication No. 2013-0059835A1.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, can also beprovided in combination in a single embodiment (while the embodimentsare intended to be combined as if written in multiply dependent form).Conversely, various features of the invention which are, for brevity,described in the context of a single embodiment, can also be providedseparately or in any suitable subcombination.

The salts and compounds described herein can be asymmetric (e.g., havingone or more stereocenters). If no stereochemistry is indicated, then allstereoisomers, such as enantiomers and diastereomers, are intendedunless otherwise indicated by the structure or name. Salts and compoundsof the present application that contain asymmetrically substitutedcarbon atoms can be isolated in optically active or racemic forms.Methods on how to prepare optically active forms from optically inactivestarting materials are known in the art, such as by resolution ofracemic mixtures or by stereoselective synthesis. Many geometric isomersof olefins, C═N double bonds, and the like can also be present in thesalts and compounds described herein, and all such stable isomers arecontemplated in the present application. Cis and trans geometric isomersof the salts and compounds of the present application are described andmay be isolated as a mixture of isomers or as separated isomeric forms.

Resolution of racemic mixtures of salts and compounds can be carried outby any of numerous methods known in the art. An example method includesfractional recrystallization using a chiral resolving acid which is anoptically active, salt-forming organic acid. Suitable resolving agentsfor fractional recrystallization methods are, for example, opticallyactive acids, such as the D and L forms of tartaric acid,diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malicacid, lactic acid or the various optically active camphorsulfonic acidssuch as 0-camphorsulfonic acid. Other resolving agents suitable forfractional crystallization methods include stereoisomerically pure formsof α-methylbenzylamine (e.g., S and R forms, or diastereomerically pureforms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine,cyclohexylethylamine, 1,2-diaminocyclohexane, and the like.

Resolution of racemic mixtures can also be carried out by elution on acolumn packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

Salts and compounds of the invention can also include all isotopes ofatoms occurring in the intermediates or final salts or compounds.Isotopes include those atoms having the same atomic number but differentmass numbers. For example, isotopes of hydrogen include tritium anddeuterium.

In some embodiments, the compounds or salts can be found together withother substances such as water and solvents (e.g. hydrates and solvates)or can be isolated.

In some embodiments, the compounds described herein, or salts thereof(e.g., the hydrochloric acid salt of the compound of Formula I), aresubstantially isolated. By “substantially isolated” is meant that thecompound is at least partially or substantially separated from theenvironment in which it was formed or detected. Partial separation caninclude, for example, a composition enriched in the compounds of theinvention. Substantial separation can include compositions containing atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 97%, or atleast about 99% by weight of the compounds of the invention, or saltthereof. Methods for isolating compounds and their salts are routine inthe art.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As will be appreciated, the compounds provided herein, including saltsthereof, can be prepared using known organic synthesis techniques andcan be synthesized according to any of numerous possible syntheticroutes. The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., 1H or 13C), infrared spectroscopy, orspectrophotometry (e.g., UV-visible); or by chromatography such as highperformance liquid chromatography (HPLC) or thin layer chromatography(TLC) or other related techniques.

As used herein, the term “reacting” is used as known in the art andgenerally refers to the bringing together of chemical reagents in such amanner so as to allow their interaction at the molecular level toachieve a chemical or physical transformation. In some embodiments, thereacting involves two reagents, wherein one or more equivalents ofsecond reagent are used with respect to the first reagent. The reactingsteps of the processes described herein can be conducted for a time andunder conditions suitable for preparing the identified product.

The reactions of the processes described herein can be carried out insuitable solvents which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,e.g., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Suitable solvents can include halogenated solvents such as carbontetrachloride, bromodichloromethane, dibromochloromethane, bromoform,chloroform, bromochloromethane, dibromomethane, butyl chloride,dichloromethane, tetrachloroethylene, trichloroethylene,1,1,1-trichloroethane, 1,1,2-trichloroethane, 1,1-dichloroethane,2-chloropropane, 1,2-dichloroethane, 1,2-dibromoethane,hexafluorobenzene, 1,2,4-trichlorobenzene, 1,2-dichlorobenzene,chlorobenzene, fluorobenzene, mixtures thereof and the like.

Suitable ether solvents include: dimethoxymethane, tetrahydrofuran,1,3-dioxane, 1,4-dioxane, furan, diethyl ether, ethylene glycol dimethylether, ethylene glycol diethyl ether, diethylene glycol dimethyl ether,diethylene glycol diethyl ether, triethylene glycol dimethyl ether,anisole, t-butyl methyl ether, mixtures thereof and the like.

Suitable protic solvents can include, by way of example and withoutlimitation, water, methanol, ethanol, 2-nitroethanol, 2-fluoroethanol,2,2,2-trifluoroethanol, ethylene glycol, 1-propanol, 2-propanol,2-methoxyethanol, 1-butanol, 2-butanol, i-butyl alcohol, t-butylalcohol, 2-ethoxyethanol, diethylene glycol, 1-, 2-, or 3-pentanol,neo-pentyl alcohol, t-pentyl alcohol, diethylene glycol monomethylether, diethylene glycol monoethyl ether, cyclohexanol, benzyl alcohol,phenol, or glycerol.

Suitable aprotic solvents can include, by way of example and withoutlimitation, tetrahydrofuran (THF), N,N-dimethylformamide (DMF),N,N-dimethylacetamide (DMA),1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU),1,3-dimethyl-2-imidazolidinone (DMI), N methylpyrrolidinone (NMP),formamide, N-methylacetamide, N-methylformamide, acetonitrile, dimethylsulfoxide, propionitrile, ethyl formate, methyl acetate,hexachloroacetone, acetone, ethyl methyl ketone, ethyl acetate,sulfolane, N,N-dimethylpropionamide, tetramethylurea, nitromethane,nitrobenzene, or hexamethylphosphoramide.

Suitable hydrocarbon solvents include benzene, cyclohexane, pentane,hexane, toluene, cycloheptane, methylcyclohexane, heptane, ethylbenzene,m-, o-, or p-xylene, octane, indane, nonane, or naphthalene.

The reactions of the processes described herein can be carried out atappropriate temperatures which can be readily determined by the skilledartisan. Reaction temperatures will depend on, for example, the meltingand boiling points of the reagents and solvent, if present; thethermodynamics of the reaction (e.g., vigorously exothermic reactionsmay need to be carried out at reduced temperatures); and the kinetics ofthe reaction (e.g., a high activation energy barrier may need elevatedtemperatures).

The expressions, “ambient temperature” and “room temperature” or “rt” asused herein, are understood in the art, and refer generally to atemperature, e.g. a reaction temperature, that is about the temperatureof the room in which the reaction is carried out, for example, atemperature from about 20° C. to about 30° C.

The reactions of the processes described herein can be carried out inair or under an inert atmosphere. Typically, reactions containingreagents or products that are substantially reactive with air can becarried out using air-sensitive synthetic techniques that are well knownto the skilled artisan.

Methods of Use

The salts and compounds of the invention can modulate activity of one ormore of various kinases including, for example, phosphoinositide3-kinases (PI3Ks). The term “modulate” is meant to refer to an abilityto increase or decrease the activity of one or more members of the PI3Kfamily. Accordingly, the salts and compounds of the invention can beused in methods of modulating a PI3K by contacting the PI3K with any oneor more of the salts, compounds or compositions described herein. Insome embodiments, salts and compounds of the present application can actas inhibitors of one or more PI3Ks. In further embodiments, the saltsand compounds of the invention can be used to modulate activity of aPI3K in an individual in need of modulation of the receptor byadministering a modulating amount of a compound of the invention, or apharmaceutically acceptable salt thereof. In some embodiments,modulating is inhibiting.

Given that cancer cell growth and survival is impacted by multiplesignaling pathways, the present application is useful for treatingdisease states characterized by drug resistant kinase mutants. Inaddition, different kinase inhibitors, exhibiting different preferencesin the kinases which they modulate the activities of, may be used incombination. This approach could prove highly efficient in treatingdisease states by targeting multiple signaling pathways, reduce thelikelihood of drug-resistance arising in a cell, and reduce the toxicityof treatments for disease.

Kinases to which the present salts and compounds bind and/or modulate(e.g., inhibit) include any member of the PI3K family. In someembodiments, the PI3K is PI3Kα, PI3Kβ, PI3Kδ, or PI3Kγ. In someembodiments, the PI3K is PI3Kδ or PI3Kγ. In some embodiments, the PI3Kis PI3Kδ. In some embodiments, the PI3K is PI3Kγ. In some embodiments,the PI3K includes a mutation. A mutation can be a replacement of oneamino acid for another, or a deletion of one or more amino acids. Insuch embodiments, the mutation can be present in the kinase domain ofthe PI3K.

In some embodiments, more than one salt or compound of the invention isused to inhibit the activity of one kinase (e.g., PI3Kδ or PI3Kγ).

In some embodiments, more than one salt or compound of the invention isused to inhibit more than one kinase, such as at least two kinases(e.g., PI3Kδ and PI3Kγ).

In some embodiments, one or more of the salts or compounds is used incombination with another kinase inhibitor to inhibit the activity of onekinase (e.g., PI3Kδ or PI3Kγ).

In some embodiments, one or more of the salts or compounds is used incombination with another kinase inhibitor to inhibit the activities ofmore than one kinase (e.g., PI3Kδ or PI3Kγ), such as at least twokinases.

The salts and compounds of the invention can be selective. By“selective” is meant that the compound binds to or inhibits a kinasewith greater affinity or potency, respectively, compared to at least oneother kinase. In some embodiments, the salts and compounds of theinvention are selective inhibitors of PI3Kδ or PI3Kγ over PI3Kα and/orPI3Kβ. In some embodiments, the salts and compounds of the invention areselective inhibitors of PI3Kδ (e.g., over PI3Kα, PI3Kβ and PI3Kγ). Insome embodiments, the salts and compounds of the invention are selectiveinhibitors of PI3Kδ (e.g., over PI3Kα, PI3Kβ and PI3Kγ). In someembodiments, selectivity can be at least about 2-fold, 5-fold, 10-fold,at least about 20-fold, at least about 50-fold, at least about 100-fold,at least about 200-fold, at least about 500-fold or at least about1000-fold. Selectivity can be measured by methods routine in the art. Insome embodiments, selectivity can be tested at the K_(m) ATPconcentration of each enzyme. In some embodiments, the selectivity ofsalts and compounds of the invention can be determined by cellularassays associated with particular PI3K kinase activity.

Another aspect of the present application pertains to methods oftreating a kinase (such as PI3K)-associated disease or disorder in anindividual (e.g., patient) by administering to the individual in need ofsuch treatment a therapeutically effective amount or dose of one or moresalts or compounds of the present application or a pharmaceuticalcomposition thereof. A PI3K-associated disease can include any disease,disorder or condition that is directly or indirectly linked toexpression or activity of the PI3K, including overexpression and/orabnormal activity levels. In some embodiments, the disease can be linkedto Akt (protein kinase B), mammalian target of rapamycin (mTOR), orphosphoinositide-dependent kinase 1 (PDK1). In some embodiments, themTOR-related disease can be inflammation, atherosclerosis, psoriasis,restenosis, benign prostatic hypertrophy, bone disorders, pancreatitis,angiogenesis, diabetic retinopathy, atherosclerosis, arthritis,immunological disorders, kidney disease, or cancer. A PI3K-associateddisease can also include any disease, disorder or condition that can beprevented, ameliorated, or cured by modulating PI3K activity. In someembodiments, the disease is characterized by the abnormal activity ofPI3K. In some embodiments, the disease is characterized by mutant PI3K.In such embodiments, the mutation can be present in the kinase domain ofthe PI3K.

Examples of PI3K-associated diseases include immune-based diseasesinvolving the system including, for example, rheumatoid arthritis,allergy, asthma, glomerulonephritis, lupus, or inflammation related toany of the above.

Further examples of PI3K-associated diseases include cancers such asbreast, prostate, colon, endometrial, brain, bladder, skin, uterus,ovary, lung, pancreatic, renal, gastric, or hematological cancer.

Further examples of PI3K-associated diseases include lung diseases suchas acute lung injury (ALI) and adult respiratory distress syndrome(ARDS).

Further examples of PI3K-associated diseases include osteoarthritis,restenosis, atherosclerosis, bone disorders, arthritis, diabeticretinopathy, psoriasis, benign prostatic hypertrophy, inflammation,angiogenesis, pancreatitis, kidney disease, inflammatory bowel disease,myasthenia gravis, multiple sclerosis, or Sjögren's syndrome, and thelike.

Further examples of PI3K-associated diseases include idiopathicthrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AIHA),vasculitis, systemic lupus erythematosus, lupus nephritis, pemphigus,membranous nephropathy, chronic lymphocytic leukemia (CLL), Non-Hodgkinlymphoma (NHL), hairy cell leukemia, Mantle cell lymphoma, Burkittlymphoma, small lymphocytic lymphoma, follicular lymphoma,lymphoplasmacytic lymphoma, extranodal marginal zone lymphoma, activatedB-cell like (ABC) diffuse large B cell lymphoma, or germinal center Bcell (GCB) diffuse large B cell lymphoma.

In some embodiments, the disease is selected from idiopathicthrombocytopenic purpura (ITP), autoimmune hemolytic anemia, vasculitis,systemic lupus erythematosus, lupus nephritis, pemphigus, autoimmunehemolytic anemia (AIHA), membranous nephropathy, chronic lymphocyticleukemia (CLL), Non-Hodgkin lymphoma (NHL), hairy cell leukemia, Mantlecell lymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicularlymphoma, lymphoplasmacytic lymphoma, extranodal marginal zone lymphoma,Hodgkin's lymphoma, Waldenstrom's macroglobulinemia, prolymphocyticleukemia, acute lymphoblastic leukemia, myelofibrosis, mucosa-associatedlymphatic tissue (MALT) lymphoma, B-cell lymphoma, mediastinal (thymic)large B-cell lymphoma, lymphomatoid granulomatosis, splenic marginalzone lymphoma, primary effusion lymphoma, intravascular large B-celllymphoma, plasma cell leukemia, extramedullary plasmacytoma, smoulderingmyeloma (aka asymptomatic myeloma), monoclonal gammopathy ofundetermined significance (MGUS) and B cell lymphoma.

In some embodiments, the method is a method of treating idiopathicthrombocytopenic purpura (ITP) selected from relapsed ITP and refractoryITP.

In some embodiments, the method is a method of treating vasculitisselected from Behçet's disease, Cogan's syndrome, giant cell arteritis,polymyalgia rheumatica (PMR), Takayasu's arteritis, Buerger's disease(thromboangiitis obliterans), central nervous system vasculitis,Kawasaki disease, polyarteritis nodosa, Churg-Strauss syndrome, mixedcryoglobulinemia vasculitis (essential or hepatitis C virus(HCV)-induced), Henoch-Schönlein purpura (HSP), hypersensitivityvasculitis, microscopic polyangiitis, Wegener's granulomatosis, andanti-neutrophil cytoplasm antibody associated (ANCA) systemic vasculitis(AASV).

In some embodiments, the method is a method of treating non-Hodgkinlymphoma (NHL) selected from relapsed NHL, refractory NHL, and recurrentfollicular NHL.

In some embodiments, the method is a method of treating B cell lymphoma,wherein said B cell lymphoma is diffuse large B-cell lymphoma (DLBCL).

In some embodiments, the method is a method of treating B cell lymphoma,wherein said B cell lymphoma is activated B-cell like (ABC) diffuselarge B cell lymphoma, or germinal center B cell (GCB) diffuse large Bcell lymphoma.

In some embodiments, said disease is osteoarthritis, restenosis,atherosclerosis, bone disorders, arthritis, diabetic retinopathy,psoriasis, benign prostatic hypertrophy, inflammation, angiogenesis,pancreatitis, kidney disease, inflammatory bowel disease, myastheniagravis, multiple sclerosis, or Sjögren's syndrome.

In some embodiments, said disease is rheumatoid arthritis, allergy,asthma, glomerulonephritis, lupus, or inflammation related to any of theaforementioned.

In some embodiments, said lupus is systemic lupus erythematosus or lupusnephritis.

In some embodiments, said disease is breast cancer, prostate cancer,colon cancer, endometrial cancer, brain cancer, bladder cancer, skincancer, cancer of the uterus, cancer of the ovary, lung cancer,pancreatic cancer, renal cancer, gastric cancer, or a hematologicalcancer.

In some embodiments, said hematological cancer is acute myeloblasticleukemia or chronic myeloid leukemia.

In some embodiments, said hematological cancer is lymphoid malignanciesof B-cell origin including, indolent/aggressive B-cell non-Hodgkin'slymphoma (NHL), and Hodgkin's lymphoma (HL).

In some embodiments, said disease is acute lung injury (ALI) or adultrespiratory distress syndrome (ARDS).

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a PI3K with a compound of the invention includesthe administration of a compound of the present application to anindividual or patient, such as a human, having a PI3K, as well as, forexample, introducing a compound of the invention into a samplecontaining a cellular or purified preparation containing the PI3K.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician. In some embodiments, the dosage ofthe compound, or a pharmaceutically acceptable salt thereof,administered to a patient or individual is about 1 mg to about 2 g,about 1 mg to about 1000 mg, about 1 mg to about 500 mg, about 1 mg toabout 100 mg, about 1 mg to 50 mg, or about 50 mg to about 500 mg.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) preventing the disease; for example, preventing a disease,condition or disorder in an individual who may be predisposed to thedisease, condition or disorder but does not yet experience or displaythe pathology or symptomatology of the disease; (2) inhibiting thedisease; for example, inhibiting a disease, condition or disorder in anindividual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder (i.e., arrestingfurther development of the pathology and/or symptomatology); and (3)ameliorating the disease; for example, ameliorating a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,reversing the pathology and/or symptomatology) such as decreasing theseverity of disease.

Combination Therapies

One or more additional pharmaceutical agents such as, for example,chemotherapeutics, anti-inflammatory agents, steroids,immunosuppressants, as well as Bcr-Abl, Flt-3, EGFR, HER2, JAK (e.g.,JAK1 or JAK2), c-MET, VEGFR, PDGFR, cKit, IGF-1R, RAF, FAK, Akt mTOR,PIM, and AKT (e.g., AKT1, AKT2, or AKT3) kinase inhibitors such as, forexample, those described in WO 2006/056399, or other agents such as,therapeutic antibodies can be used in combination with the salts orcompounds of the present application for treatment of PI3K-associateddiseases, disorders or conditions. The one or more additionalpharmaceutical agents can be administered to a patient simultaneously orsequentially.

In some embodiments, the additional pharmaceutical agent is a JAK1and/or JAK2 inhibitor. In some embodiments, the present applicationprovides a method of treating a disease described herein (e.g., a B cellmalignancy, such as diffuse B-cell lymphoma) in a patient comprisingadministering to the patient a compound described herein, or apharmaceutically acceptable salt thereof, and a JAK1 and/or JAK2inhibitor. The B cell malignancies can include those described hereinand in U.S. Ser. No. 61/976,815, filed Apr. 8, 2014.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is a compound ofTable 1, or a pharmaceutically acceptable salt thereof. The compounds inTable 1 are selective JAK1 inhibitors (selective over JAK2, JAK3, andTYK2). The IC₅₀s obtained by the method of Assay A at 1 mM ATP are shownin Table 1.

TABLE 1 JAK1 IC₅₀ JAK2/ # Prep. Name Structure (nM) JAK1 1 U.S. 2014/0121198, (Example 20) ((2R,5S)-5-{2-[(1R)-1- hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2- b]pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile

++ >10 2 U.S. 2014/ 0343030, (Example 7) 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl-1H,1'H- 4,4′-bipyrazol-1- yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2- trifluoro-1- methylethyl]benzamide

+++ >10 3 U.S. 2010/ 0298334 (Example 2)^(a) 3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4- (7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile

+ >10 4 U.S. 2010/ 0298334 (Example 13c) 3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3- yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]propanenitrile

+ >10 5 U.S. 2011/ 0059951 (Example 12) 4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4- yl)-1H-pyrazol-1- yl]propyl}piperazin-1-yl)carbonyl]-3- fluorobenzonitrile

+ >10 6 U.S. 2011/ 0059951 (Example 13) 4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4- yl)-1H-pyrrol-1- yl]propyl}piperazin-1-yl)carbonyl]-3- fluorobenzonitrile

+ >10 7 U.S. 2011/ 0224190 (Example 1) {1-{1-[3-Fluoro-2-(trifluoromethyl) isonicotinoyl] piperidin-4-yl}-3-[4- (7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 8 U.S. 2011/ 0224190 (Example 154) 4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-y])-1H- pyrazol-1-yl]azetidin-1-yl}-N-[4-fluoro-2- (trifluoromethyl)phenyl] piperidine-1-carboxamide

+ >10 9 U.S. 2011/ 0224190 (Example 85) [3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]-1-(1-{[2-(trifluoromethyl)pyrimidin- 4-yl]carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile

+ >10 10 U.S. 2012/ 0149681 (Example 7b) [trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4- yl)-1H-pyrazol-1-yl]-3-(4- {[2-(trifluoromethyl)pyrimidin- 4-yl]carbonyl}piperazin-1-yl)cyclobutyl]acetonitrile

+ >10 11 U.S. 2012/ 0149681 (Example 157) {trans-3-(4-{[4-[(3-hydroxyazetidin-1- yl)methyl]-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 12 U.S. 2012/ 0149681 (Example 161) {trans-3-(4-{[4-{[(2S)-2-(hydroxymethyl)pyrrolidin- 1-yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 13 U.S. 2012/ 0149681 (Example 162) {trans-3-(4-{[4-{[(2R)-2-(hydroxymethyl)pyrrolidin- 1-yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 14 U.S. 2012/ 0149682 (Example 20)^(b) 4-(4-{3-[(dimethylamino)methyl]- 5- fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile

+ >10 15 U.S. 2013/ 0018034 (Example 18) 5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine-2- carboxamide

+ >10 16 U.S. 2013/ 0018034 (Example 28) 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)- 2,2,2-trifluoro-1- methylethyl]benzamide

+ >10 17 U.S. 2013/ 0018034 (Example 34) 5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin- 4-yl)-1H-pyrazol-1- yl]azetidin-1-yl}-N-isopropylpyrazine-2- carboxamide

+ >10 18 U.S. 2013/ 0045963 (Example 45) {1-(cis-4-{[6-(2-hydroxyethyl)-2- (trifluoromethyl)pyrimidin- 4-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-3-yl)acetonitrile

+ >10 19 U.S. 2013/ 0045963 (Example 65) {1-(cis-4-{[4-[(ethylamino)methyl]-6- (trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4- (7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 20 U.S. 2013/ 0045963 (Example 69) {1-(cis-4-{[4-(1-hydroxy-1-methylethyl)-6- (trifluoromethyl)pyridin-2- yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]azetidin-3-yl}acetonitrile

+ >10 21 U.S. 2013/ 0045963 (Example 95) {1-(cis-4-{[4-{[(3R)-3-hydroxypyrrolidin-1- yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4- (7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 22 U.S. 2013/ 0045963 (Example 95) {1-(cis-4-{[4-{[(3S)-3-hydroxypyrrolidin-1- yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4- (7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3- yl}acetonitrile

+ >10 23 U.S. 2014/ 0005166 (Example 1) {trans-3-(4-{[4-({[(1S)-2-hydroxy-1- methylethyl]amino} methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 24 U.S. 2014/ 0005166 (Example 14) {trans-3-(4-{[4-({[(2R)-2-hydroxypropyl]amino} methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 25 U.S. 2014/ 0005166 (Example 15) {trans-3-(4-{[4-({[(2S)-2-hydroxypropyl]amino} methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1- [4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1- yl]cyclobutyl}acetonitrile

+ >10 26 U.S. 2014/ 0005166 (Example 20) {trans-3-(4-{[4-(2-hydroxyethyl)-6- (trifluoromethyl)pyridin-2- yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H- pyrazol-1-yl]cyclobutyl}acetonitrile

+ >10 + means <10 nM ++ means ≤100 nM +++ means ≤300 nM ^(a)Data forenantiomer 1 ^(b)Data for enantiomer 2

In some embodiments, the inhibitor of JAK1 and/or JAK2 is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileadipic acid salt.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide, or a pharmaceuticallyacceptable salt thereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is selected from(R)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(R)-3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(R)-4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,(R)-4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,or(R)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile,(S)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(S)-3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(S)-4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,(S)-4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,(S)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile;and pharmaceutically acceptable salts of any of the aforementioned.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is selected fromthe compounds of US Patent Publ. No. 2010/0298334, filed May 21, 2010,US Patent Publ. No. 2011/0059951, filed Aug. 31, 2010, US Patent Publ.No. 2011/0224190, filed Mar. 9, 2011, US Patent Publ. No. 2012/0149681,filed Nov. 18, 2011, US Patent Publ. No. 2012/0149682, filed Nov. 18,2011, US Patent Publ. 2013/0018034, filed Jun. 19, 2012, US Patent Publ.No. 2013/0045963, filed Aug. 17, 2012, and US Patent Publ. No.2014/0005166, filed May 17, 2013, each of which is incorporated hereinby reference in its entirety.

Example antibodies for use in combination therapy include but are notlimited to trastuzumab (e.g. anti-HER2), ranibizumab (e.g. anti-VEGF-A),bevacizumab (Avastin™, e.g. anti-VEGF), panitumumab (e.g. anti-EGFR),cetuximab (e.g. anti-EGFR), rituximab (Rituxan™, anti-CD20) andantibodies directed to c-MET.

One or more of the following agents may be used in combination with thesalts or compounds of the present application and are presented as anon-limiting list: a cytostatic agent, cisplatin, doxorubicin, taxotere,taxol, etoposide, irinotecan, camptostar, topotecan, paclitaxel,docetaxel, epothilones, tamoxifen, 5-fluorouracil, methoxtrexate,temozolomide, cyclophosphamide, SCH 66336, R115777, L778,123, BMS214662, Iressa, Tarceva, antibodies to EGFR, Gleevec™, intron, ara-C,adriamycin, cytoxan, gemcitabine, Uracil mustard, Chlormethine,Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, oxaliplatin, leucovirin,ELOXATIN™, Pentostatine, Vinblastine, Vincristine, Vindesine, Bleomycin,Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin,Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase, Teniposide17.alpha.-Ethinylestradiol, Diethylstilbestrol, Testosterone,Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone,Megestrolacetate, Methylprednisolone, Methyltestosterone, Prednisolone,Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide,Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide,Toremifene, goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine,Procarbazine, Mitotane, Mitoxantrone, Levamisole, Navelbene,Anastrazole, Letrazole, Capecitabine, Reloxafine, Droloxafine,Hexamethylmelamine, Avastin, herceptin, Bexxar, Velcade, Zevalin,Trisenox, Xeloda, Vinorelbine, Porfimer, Erbitux, Liposomal, Thiotepa,Altretamine, Melphalan, Trastuzumab, Lerozole, Fulvestrant, Exemestane,Fulvestrant, Ifosfomide, Rituximab, C225, Campath, Clofarabine,cladribine, aphidicolon, rituxan, sunitinib, dasatinib, tezacitabine,Sml1, fludarabine, pentostatin, triapine, didox, trimidox, amidox, 3-AP,MDL-101,731, bendamustine (Treanda), ofatumumab, and GS-1101 (also knownas CAL-101).

Example chemotherapeutics include proteosome inhibitors (e.g.,bortezomib), thalidomide, revlimid, and DNA-damaging agents such asmelphalan, doxorubicin, cyclophosphamide, vincristine, etoposide,carmustine, and the like.

Example steroids include coriticosteroids such as dexamethasone orprednisone.

Example Bcr-Abl inhibitors include the compounds, and pharmaceuticallyacceptable salts thereof, of the genera and species disclosed in U.S.Pat. No. 5,521,184, WO 04/005281, and U.S. Ser. No. 60/578,491.

Example suitable Flt-3 inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 03/037347, WO03/099771, and WO 04/046120.

Example suitable RAF inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO05/028444.

Example suitable FAK inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 04/080980, WO04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402.

Example suitable mTOR inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 2011/025889.

In some embodiments, the salts and compounds of the invention can beused in combination with one or more other kinase inhibitors includingimatinib, particularly for treating patients resistant to imatinib orother kinase inhibitors.

In some embodiments, the salts and compounds of the invention can beused in combination with a chemotherapeutic in the treatment of cancer,such as multiple myeloma, and may improve the treatment response ascompared to the response to the chemotherapeutic agent alone, withoutexacerbation of its toxic effects. Examples of additional pharmaceuticalagents used in the treatment of multiple myeloma, for example, caninclude, without limitation, melphalan, melphalan plus prednisone [MP],doxorubicin, dexamethasone, and Velcade (bortezomib). Further additionalagents used in the treatment of multiple myeloma include Bcr-Abl, Flt-3,RAF and FAK kinase inhibitors. Additive or synergistic effects aredesirable outcomes of combining a PI3K inhibitor of the presentapplication with an additional agent. Furthermore, resistance ofmultiple myeloma cells to agents such as dexamethasone may be reversibleupon treatment with the PI3K inhibitor of the present application. Theagents can be combined with the present compound in a single orcontinuous dosage form, or the agents can be administered simultaneouslyor sequentially as separate dosage forms.

In some embodiments, a corticosteroid such as dexamethasone isadministered to a patient in combination with the salts and compounds ofthe invention where the dexamethasone is administered intermittently asopposed to continuously.

In some further embodiments, combinations of the salts and compounds ofthe invention with other therapeutic agents can be administered to apatient prior to, during, and/or after a bone marrow transplant or stemcell transplant.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the compounds and salts of theinvention can be administered in the form of pharmaceuticalcompositions. These compositions can be prepared in a manner well knownin the pharmaceutical art, and can be administered by a variety ofroutes, depending upon whether local or systemic treatment is desiredand upon the area to be treated. Administration may be topical(including transdermal, epidermal, ophthalmic and to mucous membranesincluding intranasal, vaginal and rectal delivery), pulmonary (e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, the compound or salt of the invention (e.g.,the hydrochloric acid salt of the compound Formula I), in combinationwith one or more pharmaceutically acceptable carriers (excipients). Insome embodiments, the composition is suitable for topicaladministration. In making the compositions of the invention, the activeingredient is typically mixed with an excipient, diluted by an excipientor enclosed within such a carrier in the form of, for example, acapsule, sachet, paper, or other container. When the excipient serves asa diluent, it can be a solid, semi-solid, or liquid material, which actsas a vehicle, carrier or medium for the active ingredient. Thus, thecompositions can be in the form of tablets, pills, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,aerosols (as a solid or in a liquid medium), ointments containing, forexample, up to 10% by weight of the active compound, soft and hardgelatin capsules, suppositories, sterile injectable solutions, andsterile packaged powders.

In preparing a formulation, the active compound or salt can be milled toprovide the appropriate particle size prior to combining with the otheringredients. If the active compound or salt is substantially insoluble,it can be milled to a particle size of less than 200 mesh. If the activecompound or salt is substantially water soluble, the particle size canbe adjusted by milling to provide a substantially uniform distributionin the formulation, e.g. about 40 mesh.

The compounds and salts of the invention may be milled using knownmilling procedures such as wet milling to obtain a particle sizeappropriate for tablet formation and for other formulation types. Finelydivided (nanoparticulate) preparations of the salts and compounds of theinvention can be prepared by processes known in the art, e.g., seeInternational App. No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

In some embodiments, the compositions of the invention contain fromabout 5 to about 50 mg of the active ingredient. One having ordinaryskill in the art will appreciate that this embodies compositionscontaining about 5 to about 10, about 10 to about 15, about 15 to about20, about 20 to about 25, about 25 to about 30, about 30 to about 35,about 35 to about 40, about 40 to about 45, or about 45 to about 50 mgof the active ingredient.

In some embodiments, the compositions of the invention contain about2.5, about 5, about 7.5, about 10, about 12.5, about 15, about 17.5,about 20, about 22.5, or about 25 mg of the active ingredient. In someembodiments, the compositions of the invention contain about 5 mg of theactive ingredient. In some embodiments, the compositions of theinvention contain about 10 mg of the active ingredient.

Similar dosages may be used of the compounds and salts described hereinin the methods and uses of the invention.

The active compound or salt can be effective over a wide dosage rangeand is generally administered in a pharmaceutically effective amount. Itwill be understood, however, that the amount of the compound or saltactually administered will usually be determined by a physician,according to the relevant circumstances, including the condition to betreated, the chosen route of administration, the actual compound or saltadministered, the age, weight, and response of the individual patient,the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound or salt of the present application. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present application.

The tablets or pills of the present application can be coated orotherwise compounded to provide a dosage form affording the advantage ofprolonged action. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermit the inner component to pass intact into the duodenum or to bedelayed in release. A variety of materials can be used for such entericlayers or coatings, such materials including a number of polymeric acidsand mixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compounds, salts, and compositions of thepresent application can be incorporated for administration orally or byinjection include aqueous solutions, suitably flavored syrups, aqueousor oil suspensions, and flavored emulsions with edible oils such ascottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases. Nebulized solutionsmay be breathed directly from the nebulizing device or the nebulizingdevice can be attached to a face mask, tent, or intermittent positivepressure breathing machine. Solution, suspension, or powder compositionscan be administered orally or nasally from devices which deliver theformulation in an appropriate manner.

Topical formulations can contain one or more conventional carriers. Insome embodiments, ointments can contain water and one or morehydrophobic carriers selected from, for example, liquid paraffin,polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and thelike. Carrier compositions of creams can be based on water incombination with glycerol and one or more other components, e.g.glycerinemonostearate, PEG-glycerinemonostearate and cetylstearylalcohol. Gels can be formulated using isopropyl alcohol and water,suitably in combination with other components such as, for example,glycerol, hydroxyethyl cellulose, and the like. In some embodiments,topical formulations contain at least about 0.1, at least about 0.25, atleast about 0.5, at least about 1, at least about 2, or at least about 5wt % of the compound or salt of the invention. The topical formulationscan be suitably packaged in tubes of, for example, 100 g which areoptionally associated with instructions for the treatment of the selectindication, e.g., psoriasis or other skin condition.

The amount of compound, salt, or composition administered to a patientwill vary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the compoundpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of a compound or salt of the present applicationcan vary according to, for example, the particular use for which thetreatment is made, the manner of administration of the compound or salt,the health and condition of the patient, and the judgment of theprescribing physician. The proportion or concentration of a compound orsalt of the invention in a pharmaceutical composition can vary dependingupon a number of factors including dosage, chemical characteristics(e.g., hydrophobicity), and the route of administration. For example,the compounds and salts of the invention can be provided in an aqueousphysiological buffer solution containing about 0.1 to about 10% w/v ofthe compound for parenteral administration. Some typical dose ranges arefrom about 1 μg/kg to about 1 g/kg of body weight per day. In someembodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kgof body weight per day. The dosage is likely to depend on such variablesas the type and extent of progression of the disease or disorder, theoverall health status of the particular patient, the relative biologicalefficacy of the compound selected, formulation of the excipient, and itsroute of administration. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents such as a chemotherapeutic, steroid,anti-inflammatory compound, or immunosuppressant, examples of which areprovided herein.

Kits

The present application also includes pharmaceutical kits useful, forexample, in the treatment or prevention of PI3K-associated diseases ordisorders, such as cancer, which include one or more containerscontaining a pharmaceutical composition comprising a therapeuticallyeffective amount of a compound of the invention. Such kits can furtherinclude, if desired, one or more of various conventional pharmaceuticalkit components, such as, for example, containers with one or morepharmaceutically acceptable carriers, additional containers, etc., aswill be readily apparent to those skilled in the art. Instructions,either as inserts or as labels, indicating quantities of the componentsto be administered, guidelines for administration, and/or guidelines formixing the components, can also be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results. The hydrochloric acid salt of the compound of Formula Iand the compound of Formula I have been found to be PI3K inhibitorsaccording to at least one assay described herein.

Examples

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. The examplecompounds, or salts thereof, containing one or more chiral centers wereobtained in racemate form or as isomeric mixtures, unless otherwisespecified.

General Methods

Preparatory LC-MS purifications of some of the compounds prepared wereperformed on Waters mass directed fractionation systems. The basicequipment setup, protocols, and control software for the operation ofthese systems have been described in detail in the literature. See e.g.“Two-Pump At Column Dilution Configuration for Preparative LC-MS”, K.Blom, J. Combi. Chem., 4, 295 (2002); “Optimizing Preparative LC-MSConfigurations and Methods for Parallel Synthesis Purification”, K.Blom, R. Sparks, J. Doughty, G. Everlof, T. Haque, A. Combs, J. Combi.Chem., 5, 670 (2003); and “Preparative LC-MS Purification: ImprovedCompound Specific Method Optimization”, K. Blom, B. Glass, R. Sparks, A.Combs, J. Combi. Chem., 6, 874-883 (2004). The compounds separated weretypically subjected to analytical liquid chromatography massspectrometry (LCMS) for purity analysis under the following conditions:Instrument; Agilent 1100 series, LC/MSD, Column: Waters Sunfire™ C₁₈ 5μm, 2.1×50 mm, Buffers: mobile phase A: 0.025% TFA in water and mobilephase B: acetonitrile; gradient 2% to 80% of B in 3 minutes with flowrate 2.0 mL/minute.

Some of the compounds prepared were also separated on a preparativescale by reverse-phase high performance liquid chromatography (RP-HPLC)with MS detector or flash chromatography (silica gel) as indicated inthe Examples. Typical preparative reverse-phase high performance liquidchromatography (RP-HPLC) column conditions are as follows:

pH=2 purifications: Waters Sunfire™ C₁₈ 5 μm, 19×100 mm column, elutingwith mobile phase A: 0.1% TFA (trifluoroacetic acid) in water and mobilephase B: acetonitrile; the flow rate was 30 mL/minute, the separatinggradient was optimized for each compound using the Compound SpecificMethod Optimization protocol as described in the literature [see“Preparative LCMS Purification: Improved Compound Specific MethodOptimization”, K. Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem.,6, 874-883 (2004)]. Typically, the flow rate used with the 30×100 mmcolumn was 60 mL/minute.

pH=10 purifications: Waters XBridge C₁₈ 5 μm, 19×100 mm column, elutingwith mobile phase A: 0.15% NH₄OH in water and mobile phase B:acetonitrile; the flow rate was 30 mL/minute, the separating gradientwas optimized for each compound using the Compound Specific MethodOptimization protocol as described in the literature [See “PreparativeLCMS Purification: Improved Compound Specific Method Optimization”, K.Blom, B. Glass, R. Sparks, A. Combs, J. Comb. Chem., 6, 874-883 (2004)].Typically, the flow rate used with 30×100 mm column was 60 mL/minute.

Some of the compounds prepared were also analyzed via DifferentialScanning Calorimetry (DSC). Typical DSC instrument conditions are asfollows:

TA Instrument Differential Scanning Calorimetry, Model Q200 withautosampler: 30-350° C. at 10° C./min; T-zero aluminum sample pan andlid; nitrogen gas flow at 50 mL/min.

Mettler Toledo Differential Scanning Calorimetry (DSC) 822 Instrument:40-340° C. at a heating rate of 10° C./min.

Some of the compounds prepared were also analyzed via ThermogravimetricAnalysis (TGA). Typical TGA instrument conditions are as follows:

TA Instrument Thermogravimetric Analyzer, Model Pyris: Temperature rampfrom 25° C. to 300° C. at 10° C./min; nitrogen purge gas flow at 60mL/min; TGA ceramic crucible sample holder.

TA Instrument Q500: Temperature ramp from 20° C. to 300° C. at 10°C./min.

Some of the compounds prepared were also analyzed via X-Ray PowderDiffraction (XRPD). Typical XRPD instrument conditions are as follows:

Bruker D2 PHASER X-Ray Powder Diffractometer instrument: X-ray radiationwavelength: 1.05406 Å CuKAI; x-ray power: 30 KV, 10 mA; sample powder:dispersed on a zero-background sample holder; general measurementconditions: start Angle—5 degree, Stop Angle—60 degree, Sampling—0.015degree, Scan speed—2 degree/min.

Rigaku Miniflex Powder Diffractometer: Cu at 1.054056 Å with Kβ filter;general measurement conditions: start Angle—3 degree, Stop Angle—45degree, Sampling—0.02 degree, Scan speed—2 degree/min.

Example 1. Synthesis of(R)-4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)pyrrolidin-2-one

Step 1. 1-(5-Chloro-4-fluoro-2-hydroxyphenyl)ethanone (ii)

4-Chloro-3-fluorophenol (i, 166 g, 1.11 mol) and acetyl chloride (107mL, 1.50 mol) were charged to a 5-L flask at room temperature. Thereaction mixture was stirred and turned to a clear solution while thebatch temperature was recorded to decrease to 6° C. The reaction mixturewas then heated to 60° C. for 2 h. The reaction mixture was charged withnitrobenzene (187.5 mL, 1.82 mol) and subsequently cooled to roomtemperature. Aluminum trichloride (160 g, 1.2 mmol) was then added tothe mixture in three portions (50 g, 50 g, and 60 g at 5 min intervals).The batch temperature increased to 78° C. upon completion of addition.The reaction mixture was then heated at 100-120° C. for 3 h, at whichtime HPLC analysis showed the reaction was complete. The reactionmixture was then cooled to 0° C. and charged with hexanes (0.45 L),ethyl acetate (0.55 L), and then charged slowly with 1.0 N aqueoushydrochloric acid (1.0 L) at room temperature. The addition of aqueoushydrochloride acid was exothermic and the batch temperature increasedfrom 26° C. to 60° C. The resulting mixture was stirred at roomtemperature for 20 min. The layers were separated and the organic layerwas washed sequentially with 1.0 N aqueous hydrochloric acid (2×600 mL)and water (400 mL). The organic layer was then extracted with 1.0 Naqueous sodium hydroxide solution (2×1.4 L). The combined basicsolutions were acidified to pH 2 by addition of 12 N aqueoushydrochloric acid until no further precipitate was separated. Theresulting solid was collected by filtration, washed with water and driedin the filter funnel under suction to give compound ii as a yellow solid(187.4 g, 89.5%). ¹H NMR (400 MHz, CDCl₃) δ 12.44 (d, J=1.4 Hz, 1H),7.78 (d, J=8.1 Hz, 1H), 6.77 (d, J=10.2 Hz, 1H), 2.61 (s, 3H).

Step 2. 1-(5-Chloro-4-fluoro-2-hydroxy-3-iodophenyl)ethanone (iii)

1-(5-Chloro-4-fluoro-2-hydroxyphenyl)ethanone (ii, 100.0 g, 530.3 mmol)was dissolved in acetic acid (302 mL) and N-iodosuccinimide (179.2 g,796.5 mmol) was added to the solution. The reaction mixture was stirredat from about 61° C. to about 71° C. for 2 h, at which time HPLCanalysis indicated that the reaction was complete. The reaction mixturewas then cooled to room temperature, water (613 mL) was added, and theresulting slurry was stirred at room temperature for 30 min. The productwas collected by filtration and rinsed with water to afford brownsolids. The wet product was dissolved in acetic acid (400 mL) at 60° C.Water (800 mL) was added (over 15 min) to the solution to precipitatepure product. The product was collected by filtration and washed withwater (100 mL). The product was dried on the filter funnel under suctionfor 18 h to give compound iii as a brown solid (164.8 g, 95.0% yield).¹H NMR (300 MHz, DMSO-d₆) δ 13.34 (s, 1H), 8.26 (d, J=8.4 Hz, 1H), 2.68(s, 3H).

Step 3. 1-(5-Chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone (iv)

In a 5-L three-necked round bottom flask equipped with a condenser and athermometer, 1-(5-chloro-4-fluoro-2-hydroxy-3-iodophenyl)ethanone (iii,280 g, 840 mmol) was dissolved in N,N-dimethylformamide (600 mL). Duringthe dissolution, the internal temperature dropped from 19.3° C. to 17.0°C. Iodoethane (81.2 mL, 1020 mmol) was added to the resulting mixture.Potassium carbonate (234 g, 1690 mmol) was then added over 2 min to thereaction mixture and no change in the batch temperature was observed.The reaction mixture was heated to 60° C. for 3 h, at which time HPLCanalysis indicated the reaction was complete. The reaction mixture wasallowed to cool to room temperature and the product was collected byfiltration. The solids were dissolved in a mixture of DCM (1.0 L),hexane (500 ml), and water (2.1 L). The biphasic system was stirred at20° C. for 20 min. The layers were separated and the aqueous layer wasextracted with DCM (1.0 L). The combined organic layer was washed withwater (2×250 mL) and brine (60 mL). The organic phase was separated,dried over anhydrous Na₂SO₄, filtered, and concentrated in vacuo todryness to give compound iv as a yellow solid (292 g, 94% yield). ¹H NMR(400 MHz, DMSO-d₆) δ 7.69 (d, J=8.4 Hz, 1H), 3.95 (q, J=7.0 Hz, 2H),2.62 (s, 3H), 1.49 (t, J=7.0 Hz, 3H). LCMS for C₁₀H₁₀ClFIO₂ (M+H)⁺:m/z=342.9.

Step 4.2-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)-2-methyl-1,3-dioxolane (v)

A solution of 1-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)ethanone (iv,250.0 g, 693.4 mmol) and 1,2-ethanediol (58.0 mL, 1040 mmol) in toluene(1.5 L) was treated with p-toluenesulfonic acid monohydrate (10.6 g,55.5 mmol). The reaction flask was fitted with a Dean-Stark trap and themixture was heated at reflux for 7 h. LCMS analysis indicated that thereaction mixture contained 8.3% starting material and 91.7% product. Thereaction mixture was cooled to 106° C., and additional amount of1,2-ethanediol (11.6 mL, 208 mmol) was introduced via syringe. Thereaction mixture was then heated at reflux for an additional 8 h. LCMSanalysis indicated that the reaction mixture contained 3.6% startingmaterial and 96.4% product. The reaction mixture was cooled to 106° C.,and additional 1,2-ethanediol (7.73 mL, 139 mmol) was introduced viasyringe. The reaction mixture was heated under reflux for additional15.5 h. LCMS analysis indicated that the reaction mixture contained 2.2%starting material and 97.8% product.

The reaction mixture was then cooled to 0° C. and water (200 ml) andaqueous saturated NaHCO₃ (300 ml) were added to adjust the mixture to apH of 9. DCM (200 ml) was added and the batch was stirred for 10 min.The layers were separated and the aqueous layer was extracted withtoluene (300 mL). The combined organic layer was washed sequentiallywith a mixture of water (200 ml) and aqueous saturated NaHCO₃ (200 ml),water (300 ml), brine (300 ml), dried over anhydrous Na₂SO₄, filtered,and concentrated in vacuo to dryness to provide the crude compound v aslight brown solid (268 g 100% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.59 (d,J=8.6 Hz, 1H), 4.26-3.96 (m, 4H), 3.92-3.72 (m, 2H), 1.74 (s, 3H), 1.50(t, J=7.0 Hz, 3H). LCMS for C₁₂H₁₄ClFIO₃ (M+H)⁺: m/z=387.0.

Step 5.3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)benzaldehyde(vi)

To a stirred solution of2-(5-chloro-2-ethoxy-4-fluoro-3-iodophenyl)-2-methyl-1,3-dioxolane (v,135.0 g, 349.2 mmol) (86.8% purity by HPLC with 5.5% of the ketone) inanhydrous tetrahydrofuran (300 mL) at about 0° C. to about 3° C. wasslowly added 1.3 M isopropylmagnesium chloride lithium chloride complexin THF (322.3 mL, 419.0 mmol) over 1 h. The reaction mixture was stirredat from about 0° C. to about 5° C. for 30 min. at which time LCMSanalysis indicated the iodo-magnesium exchange reaction was complete.N-Formylmorpholine (71.1 mL, 700 mmol) was then added to the reactionmixture over 1 h at from about 0° C. to about 8° C. The reaction mixturewas stirred at from about 0° C. to about 8° C. for an additional 1 h, atwhich time LCMS and HPLC analyses showed the starting material wasconsumed and a significant amount of de-iodination by-product,2-(5-chloro-2-ethoxy-4-fluorophenyl)-2-methyl-1,3-dioxolane wasobserved. The reaction was quenched with an aqueous solution of citricacid (120.8 g, 628.6 mmol) in water (1.20 L) at 0° C. The quenchedreaction mixture was then extracted with EtOAc (2×600 mL). The phaseswere readily separated. The combined organic layer was washedsequentially with water (300 ml) and brine (500 mL), dried overanhydrous Na₂SO₄, filtered, and concentrated in vacuo. The residue waspurified by flash column chromatography on silica gel with 0-10%EtOAc/hexane to give the crude product compound vi as a pale yellowsolid, which was a mixture of the desired product,3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)benzaldehyde(vi, 80 g, 80%) containing 36 mol % of the de-iodination by-product,2-(5-chloro-2-ethoxy-4-fluorophenyl)-2-methyl-1,3-dioxolane as indicatedby NMR analysis. The crude product compound vi was further purified byformation of the corresponding sodium bisulfite adduct.

Sodium bisulfite (36.91 g, 354.7 mmol) was dissolved in water (74.3 mL,4121 mmol). To a stirred solution of crude3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)benzaldehyde(vi, 80.00 g, 177.3 mmol) in ethyl acetate (256.0 mL), the freshlyprepared sodium bisulfite solution was added in one portion. Thesolution was stirred for about 10 min and precipitates were observed.The slurry was then stirred for an additional 1 h. Thealdehyde-bisulfite adduct was collected by filtration, washed with EtOAcand dried under vacuum and nitrogen atmosphere for 20 h to give a whitesolid (58.2 g, 83.6% yield). To the aldehyde-bisulfite adduct (58.2 g,148 mmol) mixed in 1.0 M aqueous sodium hydroxide (296 mL, 296 mmol) wasadded methyl t-butyl ether (600 mL) (MTBE). The reaction mixture wasstirred at room temperature for 6 min to give a clear biphasic mixture,and stirring was continued for an additional 5 min. The organic phasewas collected and the aqueous layer was extracted with MTBE (2×300 mL).The combined organic layers were washed with brine (300 mL), dried overanhydrous Na₂SO₄, filtered, and concentrated in vacuo to give purecompound vi as a white crystalline solid (31.4 g, 73.4% yield). ¹H NMR(400 MHz, CDCl₃) δ 10.27 (s, 1H), 7.78 (d, J=8.5 Hz, 1H), 4.10-3.96 (m,4H), 3.87-3.76 (m, 2H), 1.72 (s, 3H), 1.44 (t, J=7.0 Hz, 3H). LCMS forC₁₃H₁₅ClFO₄ (M+H)⁺: m/z=289.0.

Step 6.(E)-2-(5-chloro-2-ethoxy-4-fluoro-3-(2-nitrovinyl)phenyl)-2-methyl-1,3-dioxolane(vii)

Into a 5-L 4-neck round bottom flask equipped with overhead stirrer,septa, thermocouple, nitrogen inlet and condenser was charged3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)benzaldehyde(vi, 566.2 g, 1961 mmol), nitromethane (1060 mL, 19600 mmol), andglacial acetic acid (1120 mL). Next, the reaction mixture was chargedwith benzylamine (53.6 mL, 490 mmol) and the resulting mixture washeated to 60° C. and the reaction was monitored by LCMS for 5.5 h. Aninitial baseline analysis was taken at t=0. The reaction was checkedafter 2 h and 5 h. After 2 h, there was about 20% of starting materialaldehyde unreacted. After 5 h the reaction profile was as follows:starting material compound vi (<2%), intermediate imine (<4%), productcompound vii (>93%) and benzylamine Michael adduct (not detected). Atthe reaction time of 5.5 h, the reaction was deemed complete. Thereaction mixture was cooled to room temperature and diluted with ethylacetate (3.0 L). The mixture was split in half for workup due to thelarge volume involved.

Each half was treated according to the following procedure: The reactionmixture was first washed with 1.5 M NaCl in water (2×1500 mL; after eachwash the aqueous volume output increased compared to input suggestingthat acetic acid and/or nitromethane were being removed). The mixturewas then cooled to about 15° C. and washed with 4 M aqueous NaOHsolution (4×300 mL) until the aqueous extract reached pH of 8-9. Duringinitial washes the aqueous layer remained acidic, but as the aqueousbecame slightly basic during subsequent washes the mixture heated up andthe extract was dark. The layers were separated. The organic layer afterthe pH adjustment was then washed with 1.5 M sodium chloride in water(1000 mL) and water (500 mL). Emulsion and slow partition were observedduring these final washes. The organic phase was dried over anhydrousMgSO₄, filtered, and concentrated under reduced pressure. The resultingamber syrup was placed under high vacuum overnight. The syrup solidifiedand 740 g of crude product was obtained.

Heptane (1.3 L) was added to the crude product, forming a slurry, whichwas heated in a 60° C. water bath until all the solids were dissolved.The resulting solution was polish filtered into a clean 3-L 4-neck roundbottom flask equipped with an overhead stirrer and nitrogen inlet. Thefilter was rinsed with heptane (40 mL). The filtered solution was cooledto room temperature and stirred for 5 h. Solid precipitates wereobserved and the slurry was cooled to 0° C. in an ice bath for 1 h.Product was collected by filtration and the resulting wet cake waswashed with 500 mL of ice cold heptane. The product was partially driedon the filter under vacuum suction and further dried under high vacuumovernight. The filtrate and wash solution were concentrated underreduced pressure and the resulting residue was held for columnpurification.

Solids from heptane crystallization were dissolved in a small volume ofDCM and 20% EtOAc/hexane and loaded onto a column containing about 1 kgof silica gel. The column was eluted with 20% EtOAc/hexane. The desiredfractions were combined and concentrated under reduced pressure to givea yellow solid. The solid was dried under high vacuum overnight to give497 g of product compound vii as a pale yellow crystalline solid.

The concentrated filtrate and wash from the heptane crystallization wasloaded onto the same column as above using 20% EtOAc/hexane. The columnwas eluted using the same solvent system as above to remove baselineimpurities and residual acetic acid. The desired fractions were combinedand concentrated under reduced pressure to give a reddish amber oil. Theoil was placed under high vacuum and about 220 g of crude product wasobtained. This crude product was dissolved in heptane (500 mL) andseeded with a small amount of the first crop solid product. The slurrywas stirred at room temperature for 16 h and then cooled in an ice bathfor 3 h. The second crop product was collected by filtration. Theproduct was dried under high vacuum and 110 g of product was obtained asyellow solid. The total amount of product compound vii obtained was 607g (93.3% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 7.94 (s, 2H), 7.68 (d,J=8.9 Hz, 1H), 4.07-3.95 (m, 4H), 3.82-3.73 (m, 2H), 1.65 (s, 3H), 1.39(t, J=7.0 Hz, 3H). LCMS for C₁₄H₁₆ClFNO₅ (M+H)⁺: m/z=332.0.

Step 7.(R)-Dimethyl-2-(1-(3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl)-2-nitroethyl)malonate(viii)

Into a 2-L round bottom flask with a magnetic stir bar and nitrogeninlet containing(E)-2-(5-chloro-2-ethoxy-4-fluoro-3-(2-nitrovinyl)phenyl)-2-methyl-1,3-dioxolane(vii, 352.8 g, 1064 mmol) was added anhydrous tetrahydrofuran (1.06 L)and dimethyl malonate (146 mL, 1280 mmol).(1S,2S)—N,N′-dibenzylcyclohexane-1,2-diamine-dibromonickel (Evans'Catalyst, 21.4 g, 26.6 mmol), was charged to the reaction mixture. Thereaction mixture was brown in color and a homogeneous solution wasobserved. The reaction mixture was stirred at room temperature for 18.5h. The reaction was analyzed after 18.5 h by HPLC. Unreacted startingmaterial, compound vii, was present at 2%. The reaction mixture wasconcentrated under reduced pressure to remove THF and the resultingresidue was purified by flash chromatography (DCM/hexanes used forloading, 1422 g of silica gel was used for this column, and the columnwas eluted with 10% to 20% EtOAc/hexane; column fractions were monitoredby TLC using 30% EtOAc/hexane as eluent and was visualized by UV). Thedesired fractions were combined and concentrated under reduced pressure.The residue was dried under high vacuum. Over time, the syrup solidifiedto light yellow solids (503.3 g) and a sample was taken for chiral HPLCanalysis. Chiral purity was 95.7% of the desired (R)-enantiomer and 4.3%of the undesired (S)-enantiomer. Ethanol (1.0 L) was added to the solidsand the mixture was heated in a 60° C. water bath until the solids weredissolved. The solution was polish filtered into a clean 3-L 4-neckround bottom through #1 Whatman filter paper. The filtered solution wascooled to room temperature while stirring. Crystallization was observedafter 30 min of stirring and the slurry was stirred at room temperaturefor 16 h. The slurry was cooled in an ice bath for 1 h. The product wascollected by filtration and the resulting filter cake was washed withice cold ethanol (500 mL) and was partially dried on the filter. Thesolids were dried under high vacuum to give the desired product viii(377.5 g) as white crystalline solids. The chiral purity was determinedby chiral HPLC to be 100% of the desired (R)-enantiomer and 0% of theundesired (S)-enantiomer.

The filtrate and wash were combined and concentrated under reducedpressure to an oil (118.9 g). The oil was dissolved in ethanol (475.0mL) (4 mL/g) and was seeded with the first crop crystals. The resultantslurry was stirred for 16 h at room temperature and then cooled in anice bath for 5.5 h. The second crop product was isolated by filtrationand was partially dried on the filter. It was dried under high vacuum togive the second crop product (31.0 g). The chiral purity was determinedby chiral HPLC to be 98.3% of the desired (R)-enantiomer and 1.7% of theundesired (S)-enantiomer. The combined yield of the first and secondcrops of product was 408.5 g in 82.8% yield. ¹H NMR (500 MHz, DMSO-d₆) δ7.51 (d, J=9.0 Hz, 1H), 5.20-4.81 (m, 2H), 4.62 (m, 1H), 4.14-4.03 (m,2H), 4.03-3.97 (m, 2H), 3.95-3.88 (m, 1H), 3.84-3.72 (m, 2H), 3.70 (s,3H), 3.38 (s, 3H), 1.61 (s, 3H), 1.39 (t, J=6.9 Hz, 3H). LCMS forC₁₉H₂₄ClFO₉ (M+H)⁺: m/z=463.9.

Step 8. (R)-Dimethyl2-(1-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-nitroethyl)malonate(ix)

To a stirred solution of(R)-dimethyl-2-(1-(3-chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)phenyl)-2-nitroethyl)malonate(viii, 244.0 g, 526.0 mmol) in acetone (1.2 L) in a three-necked 5-Lround bottom flask, equipped with a mechanical stirrer, was added iodine(13.4 g, 52.6 mmol) at room temperature. The resulting brown solutionwas heated at 50° C. in a water bath for 30 min, at which time LCMSanalysis showed the reaction was complete. The reaction mixture wascooled to room temperature, and then quenched with a solution of sodiumthiosulfate (17.0 g, 108 mmol) in water (160 mL) to give a pale yellowclear solution. At this time, an additional amount of water (1.2 L) wascharged to the quenched solution and the resulting white slurry wasstirred at room temperature for 1 h. The solid was then collected byfiltration and re-dissolved in acetone (1.4 L) at 40° C. The solutionwas cooled to room temperature, and then additional water (1.4 L) wasadded. The resulting white slurry was stirred at room temperature for 1h. The solid was collected by filtration and washed with water (3×100ml). The solid product was dried in a filter funnel under suction with anitrogen flow for 46 h to give compound ix as a white solid (212 g, 96%yield). ¹H NMR (300 MHz, CDCl₃) δ 7.54 (d, J=8.6 Hz, 1H), 5.09-4.67 (m,3H), 4.10-3.83 (m, 3H), 3.90 (s, 3H), 3.57 (s, 3H), 2.57 (s, 3H), 1.46(t, J=7.0 Hz, 3H). LCMS for C₁₇H₂₀ClFNO₈ (M+H)⁺: m/z=420.1.

Step 9. Dimethyl2-((R)-1-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)-2-nitroethyl)malonate(x)

To a stirred solution of (3aS)-1-methyl-3,3-diphenyltetrahydro-3H-pyrrolo[1,2-c][1,3,2]oxazaborole,((S)-MeCBS, 16.39 g, 59.12 mmol, 0.1 eq.) in anhydrous THF (100 mL) in a5 L round bottom flask at room temperature were added a solution of 1.0M borane-THF complex in THF (591 mL, 591 mmol, 1 eq.) followed by borontrifluoride etherate (3.75 mL, 29.6 mmol, 0.05 eq). The resultingsolution was stirred at room temperature for 30 min. A solution ofdimethyl[(1R)-1-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)-2-nitroethyl]malonate(ix, 253.0 g, 591.2 mmol) in anhydrous THF (1.7 L) was then addeddropwise via an addition funnel over 60 min. The flask that containedthe ketone ix was rinsed with anhydrous THF (135 mL) and the solutionwas added dropwise via the addition funnel to the reaction mixture. Theresulting solution was stirred at room temperature for an additional 10min, at which time LCMS analysis showed complete conversion of theketone to the alcohol. The reaction was quenched by dropwise addition ofmethanol (71.8 mL, 1770 mmol) at 0° C. The quenched reaction mixture wasstirred at room temperature for 15 min before it was concentrated invacuo to give the crude product. The crude products of this batch and asimilar batch (using 200 g of starting material) were combined and waspurified by silica gel column chromatography using 0 to 5% of MeOH/DCMas eluent to give compound x as a white solid (437 g, 97.9% yield). ¹HNMR (300 MHz, CDCl₃) δ 7.50 (d, J=8.8 Hz, 1H), 5.13 (q, J=6.3 Hz, 1H),5.01-4.65 (m, 3H), 4.14-3.89 (m, 3H), 3.79 (s, 3H), 3.57 (s, 3H),1.57-1.42 (m, 6H). LCMS for C₁₇H₂₁ClFNNaO₈ (M+Na)⁺: m/z=444.0.

Step 10. (4R)-Methyl4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)-2-oxopyrrolidine-3-carboxylate(xi)

A 3-necked Morton round bottom flask containing dimethyl((1R)-1-{3-chloro-6-ethoxy-2-fluoro-5-[(1R)-1-hydroxyethyl]phenyl}-2-nitroethyl)malonate(x, 100.0 g, 237.1 mmol) in tetrahydrofuran (800.0 mL) and Raney Nickel(120 g after removal of water by a pipette) was fitted with a condenser,a mechanical stirrer (glass stirring rod and Teflon bearing), and twohydrogen gas balloons (after vacuum purging). The flask was placed in anoil bath at 65° C. The batch was stirred vigorously for 16 h and theballoons were periodically removed and refilled with hydrogen. A samplewas taken and analyzed by HPLC. The product, compound xi, was present at83%. There were 7.8% of uncyclized amine and 5.5% of hydroxylamineside-product in the reaction mixture. Catalyst was filtered off (caremust be taken not to allow Raney nickel to go dry to expose to air). Thefiltrate was evaporated to dryness to give 91 g of crude product as awhite foam. The crude product (91 g, 82.6% area purity) was combinedwith another similar batch of crude product (93 g, 72.8% purity) forpurification. The combined crude product (184 g) was purified by columnchromatography on silica gel (EtOAc/hexane as eluent) to give compoundxi (101.1 g, 93% HPLC purity, 59.3% crude yield). The crude material wasused for the next step without further purification. LCMS forC₁₆H₂₀ClFNO₅ (M+H)⁺: m/z=360.0.

Step 11.(4R)-4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)-2-oxopyrrolidine-3-carboxylicAcid (xii)

Into a 5 L, 4-neck round bottom flask equipped with an overhead stirrerand a nitrogen inlet was charged a solution of (4R)-methyl4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)-2-oxopyrrolidine-3-carboxylate(xi, 268 g, 581 mmol) in tetrahydrofuran (2150 mL, 26500 mmol). To thesolution, 1.0 M sodium hydroxide in water (1420 mL, 1420 mmol) wascharged. The resulting cloudy solution became clear within 1 min. Thereaction mixture was stirred overnight at room temperature. The reactionwas analyzed by LCMS after 15 h and appeared to be complete as thestarting material was not observed. The reaction mixture was cooled inan ice bath to an internal temperature of 9.5° C. and the mixture wasacidified to pH 1-2 by addition of 6.0 M aqueous hydrochloric acid(237.0 mL, 1422 mmol) via a dropping funnel over 30 min. The reactionmixture was divided in half and each half was extracted with ethylacetate (2×1 L). The combined aqueous layers were further extracted withethyl acetate (500 mL). The two organic layers were washed with brine(20% w/w NaCl/water, 2×1000 mL), dried over anhydrous MgSO₄, filtered,and concentrated to give the crude intermediate acid xii as a yellowishfoam which was used directly in the next reaction. ¹H NMR (400 MHz,DMSO-d₆) δ 12.68 (br s, 1H), 8.26 (s, 1H), 7.52 (d, J=8.0 Hz, 1H), 5.27(br s, 1H), 4.90 (q, J=6.3 Hz, 1H), 4.28 (q, J=8.8 Hz, 1H), 3.92-3.81(m, 1H), 3.76-3.65 (m, 1H), 3.57 (t, J=9.6 Hz, 1H), 3.46 (d, J=9.4 Hz,1H), 3.23 (q, J=9.5 Hz, 1H), 1.33 (t, J=6.9 Hz, 3H), 1.28 (d, J=6.4 Hz,3H). LCMS for C₁₅H₁₇ClFNNaO₅ (M+Na)⁺: m/z=368.0.

Step 12.(R)-4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)pyrrolidin-2-one(xiii)

The crude compound xii was dissolved in 1,4-dioxane (976 mL) and toluene(976 mL) and the resulting yellow solution was heated at 100° C. Thecolor of the solution became brown as the reaction progressed. Sampleswere drawn at 1 h, 2 h and 2.5 h time points. At 2.5 h, HPLC analysisshowed the acid, compound 12, was at 0.38% and the desired product,compound xiii, at 78.8%. The reaction mixture was cooled to roomtemperature and was polished filtered into a clean 3-L round-bottomflask. The solution was then concentrated under reduced pressure and theresulting residue was placed under high vacuum to give a brown foam (254g).

Acetonitrile (350 mL) was charged to the brown syrup and heated in a 65°C. water bath until dissolution was observed. The solution was cooled toroom temperature and was stirred for 16 h. Solids were separated out ofsolution. The resulting slurry was cooled in an ice bath for 1 h. Theproduct was collected by filtration and the filter cake was rinsed with400 mL of ice cold acetonitrile. The solids appeared to be hygroscopic.The solid was dissolved in DCM (2.0 L) and concentrated to a syrup whichwas placed under high vacuum to yield compound xiii as a white foam(106.4 g).

The filtrate was concentrated to a dark syrup (about 120 g) which waspurified by flash chromatography (4×330 g silica gel columns, loadedwith DCM, eluted with 50% to 100% EtOAc/hexane, and monitored by TLCusing 100% EtOAc as eluent). Fractions from chromatography wereconcentrated under reduced pressure and placed under high vacuum toyield a light brown foam (54.4 g). The foam was charged with MTBE (400mL) and MeOH (10 mL) and heated in a 56° C. water bath for 15 minutesand some solids remained. The slurry was cooled to room temperature withstirring. The slurry was filtered to remove insoluble substances. Thefiltrate was concentrated to a syrup and placed under high vacuum toyield a foam. The foam was charged with acetonitrile (72 mL, 1.5 mL/g)and heated in a 60° C. water bath until the solution becomeshomogeneous. The solution was cooled to room temperature with stirringand solids precipitated out of solution and became very thick.Additional acetonitrile (24 mL) was charged to adjust the dilution to 2mL/g. The slurry was stirred at room temperature for 16 h and thencooled in an ice bath for 1 hour. The product was collected byfiltration and rinsed with acetonitrile. Compound xiii (25 g) wasobtained as second crop. A total of 131.4 g of compound xiii wasobtained in 74.9% yield from compound xi. ¹H NMR (400 MHz, DMSO-d₆) δ7.83 (s, 1H), 7.47 (d, J=8.7 Hz, 1H), 5.24 (d, J=4.5 Hz, 1H), 4.96-4.85(m, 1H), 4.08-3.92 (m, 1H), 3.80 (qt, J=6.9, 3.5 Hz, 2H), 3.61-3.51 (m,1H), 3.25 (t, J=9.1 Hz, 1H), 2.61-2.50 (m, 1H), 2.35-2.26 (m, 1H), 1.33(t, J=7.0 Hz, 3H), 1.27 (d, J=6.4 Hz, 3H). LCMS for C₁₄H₁₈ClFNO₃ (M+H)⁺:m/z=302.0.

Example 2. Alternative synthesis of(R)-4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)pyrrolidin-2-one

Step 1. 1-(5-chloro-2-ethoxy-4-fluorophenyl)ethanone (iv-a)

1-(5-Chloro-4-fluoro-2-hydroxyphenyl)ethanone (Compound ii from Example1, Step 1, 1350 g, 7160 mmol), N,N-dimethylformamide (3.32 L),iodoethane (1340 g, 8590 mmol), and potassium carbonate (1980 g, 14300mmol) were mixed together and stirred at room temperature for 45 min.The batch temperature went up to 55° C. from 22° C. The reaction mixturewas heated to 60° C. for 1 h (the batch temperature reached 67° C. in 30min and then dropped to 60° C.). HPLC analysis indicated all startingmaterial was consumed. Water (10 L) was added in one portion (agitationwill cease if water is added in portions). The resulting slurry wasstirred at room temperature for 30 min. The product was collected byfiltration and was rinsed with water (3 L). The product dried on thefilter under vacuum for 5 days to give compound iv-a as a tan solid(1418 g). ¹H NMR (400 MHz, DMSO-d₆) δ 7.69 (d, J=8.9 Hz, 1H), 7.30 (d,J=11.6 Hz, 1H), 4.15 (q, J=7.0 Hz, 2H), 2.51 (s, 3H), 1.37 (t, J=7.0 Hz,3H).

Step 2. 2-(5-chloro-2-ethoxy-4-fluorophenyl)-2-methyl-1,3-dioxolane(v-a)

A solution of 1-(5-chloro-2-ethoxy-4-fluorophenyl)ethanone (iv-a, 1481.0g, 6836.3 mmol) was dissolved in toluene (6 L). 1,2-Ethanediol (953 mL,17100 mmol) and p-toluenesulfonic acid monohydrate (104 g, 547 mmol)were added to the solution. The reaction mixture was heated to reflux at104-110° C. with the use of a Dean-Stark trap to remove the water for17.4 h. HPLC analysis indicated 37% of starting material remainedunreacted. About 600 mL of distillate was removed and the reactionmixture was heated under reflux for additional 5 h (total 22 h). HPLCanalysis indicated no further reaction.

It was speculated that residual K₂CO₃ in the starting material compoundiv-a may have halted the reaction. Therefore, the reaction mixture wascooled to room temperature and washed with 1 N aqueous hydrochloric acid(3×6.66 L). After the aqueous acid wash, the organic layer wastransferred back to the reaction vessel. 1,2-Ethanediol (381 mL, 6840mmol) and p-toluenesulfonic acid monohydrate (104 g, 547 mmol) wereadded and the reaction mixture was heated under reflux for 16 h. HPLCanalysis indicated about 20% of starting material remained unreacted.About 100 mL of distillate was removed. 1,2-Ethanediol (380 mL, 6800mmol) was added and refluxed for 6 h (22 h total). HPLC indicated 7% ofstarting material remained unreacted. About 125 mL of distillate wasremoved. The reaction mixture was heated to reflux for 6 h (total 28 h).HPLC indicated 5.4% of starting material remained unreacted. About 125mL of distillate was removed. The reaction mixture was heated to refluxfor additional 7 h. HPLC analysis indicated 3.5% of starting materialremained unreacted. About 80 mL of distillate was removed. The reactionwas deemed complete at this point.

The reaction mixture was washed with a 1 N aqueous sodium hydroxidesolution (2×5.5 L). The first basic wash was extracted with toluene (2.1L). The combined toluene solution was washed with water (7 L) andconcentrated to give 2153 g of dark oil. HPLC analysis indicated productpurity at 93.8% with 1.90% of starting material and 0.79% of de-iodoproduct. ¹H NMR analysis indicated about 0.5 equivalent of toluene(about 256 g) remained in the product. The corrected yield of compoundv-a was 88.0%. ¹H NMR (300 MHz, CDCl₃) δ 7.51 (d, J=8.8 Hz, 1H), 6.70(d, J=11.0 Hz, 1H), 4.17-3.92 (m, 4H), 3.91-3.80 (m, 2H), 1.75 (s, 3H),1.46 (t, J=7.0 Hz, 3H).

Step 3.3-Chloro-6-ethoxy-2-fluoro-5-(2-methyl-1,3-dioxolan-2-yl)benzaldehyde(vi)

Into an oven-dried 3-L 4-neck round bottom flask equipped with anoverhead stirrer, a 500 mL addition funnel, nitrogen inlet, septa, andthermocouple was charged N,N-diisopropylamine (87.8 mL, 626 mmol) andanhydrous tetrahydrofuran (1090 mL, 13500 mmol). This solution wascooled to −72° C. and charged with 2.5 M n-butyllithium in hexanes (261mL, 652 mmol). The n-butyllithium solution was added over 18 min. Themaximum internal temperature during the addition was −65°. The dryice-acetone bath was replaced with an ice-water bath and the reactionmixture was warmed to about −5° C. to about 0° C. and held for 15 min.The reaction mixture was then cooled to −74.5° C.

To a separate 1-L round bottom flask containing2-(5-chloro-2-ethoxy-4-fluorophenyl)-2-methyl-1,3-dioxolane (v-a, 136.1g, 522.1 mmol) was added anhydrous tetrahydrofuran (456 mL) to dissolvethe solids. The resulting solution was cooled in an ice bath to about 0°C. The solution containing compound v-a was transferred to the LDAsolution over 40 minutes via a canula while maintaining the reactiontemperature between −70° C. and −72.5° C. The reaction mixture becameyellow slurry and was stirred for 37 min at −74° C.N,N-Dimethylformamide (60.6 mL, 783 mmol) was charged in one portion viaa syringe and this addition produced an exotherm from −74.5° C. to−66.5° C. The reaction was monitored by HPLC at 90 min after theaddition. The starting material was present at 2.9%. The cold bath wasremoved and the reaction mixture was warmed in ambient temperature. Thereaction mixture was sampled and analyzed after 3 h and unreactedstarting material was present at 1.5%. The reaction was deemed completeand was quenched by addition of the reaction solution to ice water (1.4L) and diluted with ethyl acetate (1.5 L). The aqueous layer wasextracted with ethyl acetate (1.5 L) and the organic layers werecombined and washed with brine (20% w/w aq. NaCl, 2×600 mL) and driedover anhydrous MgSO₄. The MgSO₄ was removed by filtration and thefiltrate was concentrated to an oil with some solids present. Thisresidue was dissolved in methylene chloride and loaded onto a pad ofsilica gel (586 g). The silica pad was eluted with 2% EtOAc/DCM(monitored by TLC using 100% DCM as eluent). The desired fractions werecollected and concentrated under reduced pressure to give a light amberoil. The oil was placed under high vacuum to give compound vi as ayellow solid (146.5 g, 95.1% yield). ¹H NMR (400 MHz, CDCl₃) δ 10.27 (s,1H), 7.78 (d, J=8.5 Hz, 1H), 4.10-3.96 (m, 4H), 3.87-3.76 (m, 2H), 1.72(s, 3H), 1.44 (t, J=7.0 Hz, 3H). LCMS for C₁₃H₁₅ClFO₄ (M+H)⁺: m/z=289.1.

Steps 4-10.(R)-4-(3-chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)pyrrolidin-2-one(xiii)

The title compound was prepared using procedures analogous to thosedescribed in Example 1, Steps 6-12.

Example 3.(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-oneHydrochloride

Step 1.(R)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethylMethanesulfonate (xiv)

(R)-4-(3-Chloro-6-ethoxy-2-fluoro-5-((R)-1-hydroxyethyl)phenyl)pyrrolidin-2-one(xiii, 172.0 g, 570.0 mmol) (consisted of 147 g at 99.83%: 0.09% chiralpurity, 99.33% chemical purity; and 25 g, 87.46%: 12.54% chiral purity,86.74% chemical purity) was dissolved in methylene chloride (860 mL).N,N-diisopropylethylamine (149 mL, 855 mmol) was added to the solutionat from about −7° C. to about 2° C. Methanesulfonyl chloride (57.4 mL,741 mmol) was added dropwise to the reaction mixture over 25 min. Thesuspension turned into a clear solution. At 30 min reaction time pointHPLC indicated the reaction was complete. This reaction mixturecontaining compound xiv was used directly in the next reaction.

Step 2.(R)-4-(3-chloro-6-ethoxy-2-fluoro-5-((S)-1-hydrazinylethyl)phenyl)pyrrolidin-2-one(xv)

At 0° C., hydrazine (178.9 mL, 5.7 mol) was added in one portionfollowed by N-methylpyrrolidinone (860 mL) to the reaction mixturecontaining compound xiv from Step 1. The reaction mixture turned cloudyand some precipitates formed. The mixture was heated to 40-57° C. undernitrogen for 90 min. HPLC indicated all the mesylate had been consumed.

The reaction mixture was cooled to room temperature and a saturatedsolution of sodium bicarbonate (28.3 g) in water (300 mL) was added. Themixture was stirred for 20 min, at which time dichloromethane (300 mL)was added. The organic layer was separated and stirred with a solutionof sodium bicarbonate (14.2 g) in water (150 mL). The aqueous layer wasextracted with dichloromethane (200 mL×2). The combined organic layerswere washed with brine (80 mL), dried over anhydrous Na₂SO₄ (311 g),concentrated, and azeotroped with toluene (250 mL) to give a colorlessN-methylpyrrolidinone solution containing compound xv which was useddirectly in the next reaction. A sample was purified for NMR analysis.¹H NMR (400 MHz, DMSO-d₆), δ 7.88 (s, 1H), 7.66 (d, J=8.5 Hz, 1H), 4.42(q, J=6.7 Hz, 1H), 4.06-3.88 (m, 2H), 3.79-3.66 (m, 1H), 3.65-3.51 (m,1H), 3.24 (t, J=8.8 Hz, 1H), 2.60-2.46 (m, 1H), 2.36-2.25 (m, 1H), 1.37(t, J=6.9 Hz, 3H), 1.26 (d, J=6.8 Hz, 3H). LCMS for C₁₄H₁₉ClFN₃O₂(M+H)⁺:m/z=316.1.

Step 3.5-Amino-1-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)-3-methyl-1H-pyrazole-4-carbonitrile(xvi)

With stirring, (1-ethoxyethylidene)malononitrile (101 g, 741 mmol) wasadded to the N-methylpyrrolidinone solution of compound xv from Step 2,in portions and the mixture was stirred at room temperature undernitrogen. After 15 min, HPLC analysis indicated 11% starting materialhydrazine, compound xv, relative to product compound xvi.N,N-Diisopropylethylamine (15 mL, 86 mmol) was added and the reactionmixture was stirred at room temperature for 15 h. HPLC analysisindicated 5.6% of starting material remained. N,N-Diisopropylethylamine(5 mL, 30 mmol) was added and the reaction mixture was stirred at roomtemperature for 5 h. HPLC indicated 5.6% starting material remained. Thereaction mixture was stirred for 2.5 days and combined with two similarbatches and worked up together.

The reaction mixtures of three batches of compound xvi were combined. Anaqueous 0.5 M sodium hydroxide solution (3.8 L) was added at 10-20° C.and stirred for 5 min. HPLC indicated that all starting material(1-ethoxyethylidene)malononitrile was consumed. Ethyl acetate (4.0 L)was added and the mixture was stirred for 15 min. The layers wereseparated. The organic layer was washed with 0.5 M sodium hydroxide inwater (2.38 L). The layers were separated. The combined aqueous layerwas extracted with ethyl acetate (2×2 L). The combined organic layerswere washed with 1.0 M aqueous hydrochloric acid (3.56 L) and the pH ofthe resulting aqueous layer was 2-3. The organic layer was washed withbrine (5 L), dried over anhydrous Na₂SO₄, concentrated, and dried underhigh vacuum for 40 h to give compound xvi as a light brown foamy solid(702.7 g). ¹H NMR (500 MHz, DMSO-d₆) δ 7.78 (s, 1H), 7.44 (d, J=8.4 Hz,1H), 6.53 (s, 2H), 5.64 (q, J=6.7 Hz, 1H), 3.96 (m, 1H), 3.74 (m, 1H),3.34 (m, 1H), 3.58 (m, 2H), 2.59-2.50 (m, 1H), 2.29 (m, 1H), 2.04 (s,3H), 1.57 (d, J=6.8 Hz, 3H), 1.37 (t, J=6.9 Hz, 3H). LCMS forC₁₉H₂₂ClFN₅O₂(M+H)⁺: m/z=406.1.

The overall yield of compound xvi three steps (mesylation,hydrazinolysis and pyrazole formation) was calculated to be 72.8% fromthe total input of compound xiii. The purity was determined by HPLC tobe about 80%. HPLC analysis indicated some product existing in the basicaqueous layer which was subsequently extracted with EtOAc (2 L), washedwith 1.0 M aqueous hydrochloric acid and brine, dried with anhydroussodium sulfate, concentrated, and dried on high vacuum pump for 40 h toafford compound xvi as a brown oil (134 g, 13.9%).

Step 4.(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one(xvii)

5-Amino-1-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)-3-methyl-1H-pyrazole-4-carbonitrile(xvi, 702.7 g, 1731 mmol) was added to a reaction vessel withformamidine acetate (1802 g, 17.31 mol) and 1,2-ethanediol (3.51 L). Thereaction mixture was heated at 102-103° C. with stirring for 18 h. Thereaction mixture was cooled to room temperature and ethyl acetate (7 L)and water (6 L) were added and the biphasic mixture was stirred for 15min. The organic layer was separated and the aqueous layer was dilutedwith additional water (4.5 L) and ethyl acetate (3 L) and stirred for 10min. The organic layer was separated. The aqueous layer was furtherextracted with ethyl acetate (2 L). The organic layers were combined andstirred with water (4.5 L). The aqueous layer was separated and theorganic layer was filtered through a pad of celite (about 1 kg). Theorganic layer was extracted with 1.0 M aqueous hydrochloric acid (7 L)by stirring the mixture for 10 min. The aqueous layer was separated. Theclear brown organic layer was stirred with additional 1.0 M aqueoushydrochloric acid (3 L) for 10 min. The aqueous layer was separated. Theaqueous acidic layers were combined and washed with toluene (500 mL).The aqueous acidic solution was cooled with an ice-water bath andmethylene chloride (4 L) was added. At 5-15° C., a solution of sodiumhydroxide (530 g) in water (530 mL) (50% NaOH solution) was added slowlyuntil to a solution pH of 11-12. Solid precipitates were observed.Additional methylene chloride (3.5 L) and methanol (300 mL) were addedand the mixture was stirred for 10-15 min. The solid product wascollected by filtration and dried on the filter under suction for 16 hto give compound xvii (289.7 g) as a brown solid. ¹H NMR (400 MHz,DMSO-d₆) δ 8.11 (s, 1H), 7.82 (s, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.30 (brs, 2H), 6.23 (q, J=7.0 Hz, 1H), 3.97 (p, J=9.2 Hz, 1H), 3.90-3.73 (m,2H), 3.57 (t, J=9.9 Hz, 1H), 3.25 (dd, J=9.2, 8.7 Hz, 1H), 2.48 (s, 3H),2.60-2.50 (m, 1H), 2.36-2.20 (m, 1H), 1.69 (d, J=7.1 Hz, 3H), 1.39 (t,J=6.9 Hz, 3H). LCMS for C₂₀H₂₃ClFN₆O₂ (M+H)⁺: m/z=433.3.

The filtrate was transferred into a separatory funnel and the organiclayer was separated. The aqueous layer was stirred with methylenechloride (5 L) and methanol (200 mL). The combined organic layer wasdried over anhydrous sodium sulfate, concentrated, dried on high vacuumpump for 16 h to give additional amount 259.3 g as a brown solid. Thetotal yield of xvii was 548.3 g in 73.2% yield.

Step 5.(R)-4-(3-((S)-1-(4-Amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-oneHydrochloride Salt (xviii)

A 1.0 M aqueous hydrochloric acid (HCl, 5.0 L, 5.0 mol) solution wasadded to(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one(xvii, 609.8 g, 1.409 mol) at room temperature. The resulting thickslurry was then heated to 50° C. to afford a clear solution. Anadditional 1.82 L of 1.0 M aqueous hydrochloric acid solution (HCl, 1.82L, 1.82 mol; total 6.82 L, 6.82 mol, 4.84 equiv) was added to the clearsolution at 50° C. and the solution was then filtered through a polishfilter at approximately 50° C. The polish filtered reaction mixture wasgradually cooled to room temperature over 2 h before it was furthercooled to 0-5° C. The reaction mixture was stirred at 0-5° C. for atleast 20 min to initiate precipitation. The resulting solids werecollected by filtration, rinsed with a portion of cold mother liquor,followed by 1.0 M aqueous hydrochloric acid (HCl, 200 mL), and dried onthe filter funnel at room temperature under suction to constant weight(in about 39 h) to afford the hydrochloric acid salt of the compound ofFormula I:(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloride (xviii, 348.7 g, 661.2 g theoretical, 52.7%) as whitecrystalline powder. ¹H NMR (400 MHz, DMSO-d₆) δ 9.39 (br s, 1H), 9.05(br s, 1H), 8.50 (s, 1H), 7.84 (s, 1H), 7.59 (d, J=8.4 Hz, 1H), 6.28 (q,J=6.9 Hz, 1H), 3.95 (m, 1H), 3.79 (m, 2H), 3.55 (m, 1H), 3.22 (m, 1H),2.59 (s, 3H), 2.55 (ddd, J=16.8, 10.3, 2.3 Hz, 1H), 2.28 (ddd, J=16.8,8.6, 1.5 Hz, 1H), 1.73 (d, J=7.0 Hz, 3H), 1.38 (t, J=6.9 Hz, 3H) ppm.¹³C NMR (100 MHz, DMSO-d₆) δ 175.3, 156.4 (J_(CF)=249.8 Hz), 153.8(J_(CF)=7.0 Hz), 152.4, 150.8, 147.3, 144.3, 131.4 (J_(CF)=3.5 Hz),127.3, 126.4 (J_(CF)=12.6 Hz), 116.1 (J_(CF)=18.4 Hz), 98.0, 72.1, 49.1,46.6, 36.0, 29.4, 21.0, 15.4, 14.6 ppm. ¹⁹F NMR (376 MHz, DMSO-d₆) δ−113.6 (d, J_(FH)=7.7 Hz) ppm. C₂₀H₂₃Cl₂FN₆O₂(MW 469.34); LCMS (EI) m/e433.2 (M+H; exact mass of xvii: 432.15). Water content by KF: 3.63% byweight; Chloride (Cl⁻) content by titration: 7.56% by weight (7.56% bytheory).

The melting/decomposition range of(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloride salt crystalline form was determined by DSC, from aninitial temperature of 30° C. to a final temperature of 350° C. using aheating rate of 10° C./min. The DSC thermogram revealed one endothermicevent with an onset at 194.37° C. and the peak at 206.55° C., as shownin FIG. 1.

The TGA thermogram showed the total weight loss of 4.3% up to 210° C.Above 210° C. the salt starts to decompose, as shown in FIG. 2.

A representative X-Ray Power Diffraction (XRPD) pattern is shown in FIG.3 and Table 2 shows the corresponding peaks and intensities.

TABLE 2 2-theta Relative Intensity 5.739 2.20% 7.133 1.20% 7.736 0.10%10.225 2.30% 11.283 99.00% 11.303 94.10% 13.666 2.90% 14.166 0.90%14.833 0.10% 15.364 3.80% 16.354 9.70% 17.136 0.50% 16.866 2.70% 17.4355.50% 17.635 3.30% 18.811 5.10% 18.898 6.60% 19.603 1.50% 20.157 1.80%20.593 0.50% 21.039 11.10% 21.308 3.80% 22.169 7.50% 23.002 11.50%24.628 6.60% 25.098 2.20% 25.66 7.00% 25.895 4.00% 27.168 3.10% 27.7928.50% 28.1 10.00% 28.464 5.50% 30.134 3.20% 31.239 13.70% 31.918 1.30%32.827 9.50% 33.818 0.70% 34.198 2.80% 35.033 2.10% 35.423 2.10% 36.2260.30% 36.676 0.90% 37.47 0.90% 37.951 0.50% 38.457 1.70% 39.055 0.20%39.968 0.20% 40.184 0.30% 40.962 0.20% 42 1.30% 42.916 2.40% 43.3730.50% 44.148 0.40% 45.29 0.30% 46.089 1.40% 47.572 0.40% 48.897 0.70%49.647 0.50% 50.589 0.30% 51.042 0.10% 51.687 0.40% 52.624 0.40% 53.2870.50% 54.104 0.20% 54.127 0.10% 54.159 0.20% 55.42 0.30% 56.821 0.10%

Example 4. Alternative Synthesis of(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-oneHydrochloride

Step 1.(R)-4-(3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one (xix)

(4R)-4-[3-Chloro-6-ethoxy-2-fluoro-5-(1-hydroxyethyl)phenyl]pyrrolidin-2-one(as a mixture of two diastereomers with R-configuration at thepyrrolidinone and R- or S-configurations at the secondary alcohol)(xiii, 16.7 g, 55.3 mmol) was dissolved in dichloromethane (167 mL). Thesolution was cooled in an ice-water bath and Dess-Martin periodinane(35.2 g, 83.0 mmol) was added in small portions. The reaction mixturewas stirred at room temperature for 2 h, at which time HPLC analysisshowed reaction completion. A solution of sodium sulfite (28 g, 220mmol) in water (70 mL) was added to the reaction mixture and the mixturewas stirred for 20 min. A 1.0 M sodium hydroxide solution was added tothe mixture and stirred for 10 min. The layers were allowed to settleand the organic layer was separated and washed sequentially with 1 Maqueous sodium hydroxide solution (66 mL) and water (60 mL). The organiclayer was dried over anhydrous sodium sulfate. The drying agent wasremoved by filtration and the filtrate was concentrated to give(R)-4-[3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl]pyrrolidin-2-one as anoil which was used in the next reaction without further purification.

Step 2. (R,E)-tert-butyl2-(1-(5-chloro-2-ethoxy-4-fluoro-3-(5-oxopyrrolidin-3-yl)phenyl)ethylidene)hydrazinecarboxylate(xx)

Crude (R)-4-[3-acetyl-5-chloro-2-ethoxy-6-fluorophenyl]pyrrolidin-2-one(compound xix from Step 1) was dissolved in methanol (60 mL) and t-butylcarbazate (8.04 g, 60.8 mmol) was added to the solution. The reactionmixture was stirred at 65° C. for 3.5 days, at which time HPLC analysisshowed reaction completion. The mixture was concentrated under reducedpressure and the residue was purified by silica gel chromatographyeluting with a mixture of 0-5% of methanol in ethyl acetate to give(R,E)-tert-butyl2-(1-(5-chloro-2-ethoxy-4-fluoro-3-(5-oxopyrrolidin-3-yl)phenyl)ethylidene)hydrazinecarboxylate(xx, 19.5 g, 85%). ¹H NMR (500 MHz, DMSO-d₆) δ 9.83 (s, 1H), 7.78 (s,1H), 7.36 (d, J=8.6 Hz, 1H), 4.07 (p, J=9.1 Hz, 1H), 3.84-3.69 (m, 2H),3.59 (t, J=9.5 Hz, 1H), 3.28 (t, J=9.5 Hz, 1H), 2.54 (m, 1H), 2.33 (m,1H), 2.14 (s, 3H), 1.46 (s, 9H), 1.25 (t, J=7.0 Hz, 3H). LCMS forC₁₉H₂₅ClFN₃NaO₄ (M+Na)⁺: m/z=436.1.

Step 3. tert-Butyl2-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)hydrazinecarboxylate(xxi)

(R,E)-tert-butyl2-(1-(5-chloro-2-ethoxy-4-fluoro-3-(5-oxopyrrolidin-3-yl)phenyl)ethylidene)hydrazinecarboxylate(xx, 0.5 g, 1.2 mmol) was dissolved in methanol (25 mL) and the solutionwas bubbled with nitrogen gas for 5 min.Bis(1,5-cyclooctadiene)rhodium(I)tetrafluoroborate (35 mg, 0.086 mmol)and(R)-(−)-1-{(S)-2-[bis(4-trifluoromethylphenyl)phosphine]ferrocenyl}ethyl-di-t-butylphosphine(64 mg, 0.094 mmol) were added to the solution and the resultingreaction mixture was bubbled with nitrogen gas for 30 min. The reactionmixture was then agitated under hydrogen gas (56 psi) pressure for 2.5days. The reaction mixture was concentrated under reduced pressure andthe resulting residue was purified by silica gel column chromatographyeluting with a mixture of methanol (0-10%) in ethyl acetate. The desiredfractions were concentrated to give tert-butyl2-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)hydrazinecarboxylate(xxi, 428 mg, 85% yield). ¹H NMR (500 MHz, DMSO-d₆) δ 8.18 (s, 1H), 7.78(s, 1H), 7.53 (d, J=8.2 Hz, 1H), 4.73 (s, 1H), 4.41 (br s, 1H), 3.98 (m,1H), 3.75 (m, 2H), 3.61 (m, 1H), 3.26 (m, 1H), 2.53 (m, 1H), 2.29 (dd,J=17.6, 8.6 Hz, 1H), 1.32 (s, 12H), 1.10 (d, J=6.5 Hz, 1H). LCMS forC₁₉H₂₇ClFN₃NaO₄ (M+Na)⁺: m/z=437.9. Chiral HPLC analysis indicated theproduct contained the desired diastereomertert-butyl-2-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)hydrazinecarboxylate (xxi) at 85.6% and the undesired diastereomertert-butyl-2-((R)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)hydrazinecarboxylat 14.3%.

Step 4.5-Amino-1-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)-3-methyl-1H-pyrazole-4-carbonitrile(xvi)

tert-Butyl2-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)hydrazinecarboxylate(xxi, 130 mg, 0.31 mmol) and p-toluenesulfonic acid monohydrate (86 mg,0.45 mmol) were added to ethanol (3 mL) and the reaction mixture washeated at 50° C. for 20 h. HPLC analysis showed there was about 88% ofunreacted starting material. Additional amount ofp-toluene sulfonic acid(86 mg, 0.45 mmol) was charged and the reaction mixture was heated to60° C. for 24 h. HPLC analysis showed complete Boc-deprotection. Thisreaction mixture was added with (1-ethoxyethylidene)malononitrile (61mg, 0.45 mmol) and N,N-diisopropylethylamine (260 μL, 1.5 mmol). Thereaction mixture was stirred at room temperature for 2 h. HPLC showedcompletion of pyrazole-ring formation. 1.0 M aqueous sodium hydroxidesolution was added to the reaction mixture and stirred for 20 min. Ethylacetate (20 mL) was added to the mixture and stirred. The biphasicmixture was allowed to settle. The ethyl acetate layer was collected andthe aqueous layer was extracted with ethyl acetate (10 mL). The combinedethyl acetate solution was added with 1M aqueous hydrochloric acid (5mL) and stirred for 15 min. The biphasic mixture was allowed to settleand the organic layer was collected and dried over anhydrous sodiumsulfate. Sodium sulfate was removed by filtration and to filtrate wasconcentrated to give5-Amino-1-((S)-1-(5-chloro-2-ethoxy-4-fluoro-3-((R)-5-oxopyrrolidin-3-yl)phenyl)ethyl)-3-methyl-1H-pyrazole-4-carbonitrile(xvi, 126 mg, quantitative yield of crude product) and was used in thenext step without further purification.

Step 5.(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one(xvii)

5-Amino-1-{(1S)-1-[5-chloro-2-ethoxy-4-fluoro-3-(5-oxopyrrolidin-3-yl)phenyl]ethyl}-3-methyl-1Hpyrazole-4-carbonitrile(xvi, 126 mg, 0.31 mmol) was added with formamidine acetate (323 mg, 3.1mmol) and 1,2-ethanediol (2 mL). The reaction mixture was heated at104-105° C. with stirring. After 18 h, HPLC analysis showed about 44% ofstarting material compound xvi remaining. The reaction mixture washeated to 115° C. for 24 h. HPLC analysis showed the reaction wascomplete. The reaction mixture was cooled to room temperature and ethylacetate (10 mL) and water (5 ml) were added. The biphasic mixture wasstirred. The layers were allowed to separate. The organic layer wascollected and the aqueous layer was extracted with ethyl acetate (5 mL).The combined ethyl acetate solution was washed with water (5 mL), driedover anhydrous sodium sulfate. Sodium sulfate was removed by filtrationand the filtrate was concentrated to a residue. The residue was purifiedby silica gel chromatography. The column was eluted with a mixture ofmethanol (0-5%) in methylene chloride. The desired fractions werecombined and evaporated to give(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one(xvii, 94 mg, 69.9% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 8.11 (s, 1H),7.82 (s, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.30 (br s, 2H), 6.23 (q, J=7.0Hz, 1H), 3.97 (p, J=9.2 Hz, 1H), 3.90-3.73 (m, 2H), 3.57 (t, J=9.9 Hz,1H), 3.25 (dd, J=9.2, 8.7 Hz, 1H), 2.48 (s, 3H), 2.60-2.50 (m, 1H),2.36-2.20 (m, 1H), 1.69 (d, J=7.1 Hz, 3H), 1.39 (t, J=6.9 Hz, 3H). LCMSfor C₂₀H₂₃ClFN₆O₂ (M+H)⁺: m/z=433.3.

Chiral HPLC analysis of the product indicated that it contained thedesired diastereomer,(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-one(xvii), at 87% and the undesired diastereomer(R)-4-(3-((R)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-oneat 13%.

Step 6.(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-oneHydrochloride

The title product was prepared according to the procedure described inExample 3, Step 5. The resulting hydrochloride salt matches well withthe material made from the synthetic process described in Example 3, inevery comparable aspect including chemical purity, chiral purity, andsolid state characteristics.

Example A1: PI3K Enzyme Assay

PI3-Kinase luminescent assay kit including lipid kinase substrate,D-myo-phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D(+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2),biotinylated I(1,3,4,5)P4, PI(3,4,5)P3 Detector Protein is purchasedfrom Echelon Biosciences (Salt Lake City, Utah). AlphaScreen™ GSTDetection Kit including donor and acceptor beads was purchased fromPerkinElmer Life Sciences (Waltham, Mass.). PI3Kδ (p110δ/p85α) ispurchased from Millipore (Bedford, Mass.). ATP, MgCl₂, DTT, EDTA, HEPESand CHAPS are purchased from Sigma-Aldrich (St. Louis, Mo.).

AlphaScreen™ Assay for PI3Kδ

The kinase reaction are conducted in 384-well REMP plate from ThermoFisher Scientific in a final volume of 40 μL. Inhibitors are firstdiluted serially in DMSO and added to the plate wells before theaddition of other reaction components. The final concentration of DMSOin the assay is 2%. The PI3K assays are carried out at room temperaturein 50 mM HEPES, pH 7.4, 5 mM MgCl₂, 50 mM NaCl, 5 mM DTT and CHAPS0.04%. Reactions are initiated by the addition of ATP, the finalreaction mixture consisted of 20 μM PIP2, 20 μM ATP, 1.2 nM PI3Kδ areincubated for 20 minutes. 10 μL of reaction mixture are then transferredto 5 μL 50 nM biotinylated I(1,3,4,5)P4 in quench buffer: 50 mM HEPES pH7.4, 150 mM NaCl, 10 mM EDTA, 5 mM DTT, 0.1% Tween-20, followed with theaddition of 10 μL AlphaScreen™ donor and acceptor beads suspended inquench buffer containing 25 nM PI(3,4,5)P3 detector protein. The finalconcentration of both donor and acceptor beads is 20 mg/ml. After platesealing, the plate are incubated in a dark location at room temperaturefor 2 hours. The activity of the product is determined on Fusion-alphamicroplate reader (Perkin-Elmer). IC₅₀ determination is performed byfitting the curve of percent control activity versus the log of theinhibitor concentration using the GraphPad Prism 3.0 software.

Example A2: PI3K Enzyme Assay

Materials:

Lipid kinase substrate, phosphoinositol-4,5-bisphosphate (PIP2), arepurchased from Echelon Biosciences (Salt Lake City, Utah). PI3K isoformsα, β, δ and γ are purchased from Millipore (Bedford, Mass.). ATP, MgCl₂,DTT, EDTA, MOPS and CHAPS are purchased from Sigma-Aldrich (St. Louis,Mo.).

The kinase reactions are conducted in clear-bottom 96-well plate fromThermo Fisher Scientific in a final volume of 24 μL. Inhibitors arefirst diluted serially in DMSO and added to the plate wells before theaddition of other reaction components. The final concentration of DMSOin the assay is 0.5%. The PI3K assays are carried out at roomtemperature in 20 mM MOPS, pH 6.7, 10 mM MgCl₂, 5 mM DTT and CHAPS0.03%. The reaction mixture is prepared containing 50 μM PIP2, kinaseand varying concentration of inhibitors. Reactions are initiated by theaddition of ATP containing 2.2 μCi [γ-³³P]ATP to a final concentrationof 1000 μM. The final concentration of PI3K isoforms α, β, δ and γ inthe assay were 1.3, 9.4, 2.9 and 10.8 nM, respectively. Reactions areincubated for 180 minutes and terminated by the addition of 100 μL of 1M potassium phosphate pH 8.0, 30 mM EDTA quench buffer. A 100 μL aliquotof the reaction solution are then transferred to 96-well MilliporeMultiScreen IP 0.45 μm PVDF filter plate (The filter plate is prewettedwith 200 μL 100% ethanol, distilled water, and 1 M potassium phosphatepH 8.0, respectively). The filter plate is aspirated on a MilliporeManifold under vacuum and washed with 18×200 μL wash buffer containing 1M potassium phosphate pH 8.0 and 1 mM ATP. After drying by aspirationand blotting, the plate is air dried in an incubator at 37° C.overnight. Packard TopCount adapter (Millipore) is then attached to theplate followed with addition of 120 μL Microscint 20 scintillationcocktail (Perkin Elmer) in each well. After the plate sealing, theradioactivity of the product is determined by scintillation counting onTopcount (Perkin-Elmer). IC₅₀ determination is performed by fitting thecurve of percent control activity versus the log of the inhibitorconcentration using the GraphPad Prism 3.0 software.

The(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloric acid salt was tested in the assay of Example A2 anddetermined to be a selective inhibitor for PI3Kδ.

The(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloric acid salt was tested in the assay of Example A2 anddetermined to be a >100 fold selective inhibitor for PI3Kδ over each ofPI3Kα, PI3Kβ, and PI3Kγ.

Example A3: PI3Kδ Scintillation Proximity Assay Materials

[γ-³³P]ATP (10 mCi/mL) was purchased from Perkin-Elmer (Waltham, Mass.).Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate(PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked(PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (SaltLake City, Utah). PI3Kδ (p110δ/p85α) was purchased from Millipore(Bedford, Mass.). ATP, MgCl₂, DTT, EDTA, MOPS and CHAPS were purchasedfrom Sigma-Aldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPAScintillation Beads was purchased from GE healthcare life sciences(Piscataway, N.J.).

The kinase reaction was conducted in polystyrene 384-well matrix whiteplate from Thermo Fisher Scientific in a final volume of 25 μL.Inhibitors were first diluted serially in DMSO and added to the platewells before the addition of other reaction components. The finalconcentration of DMSO in the assay was 0.5%. The PI3K assays werecarried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl₂, 5 mMDTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP,the final reaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi[γ-³³P] ATP, 4 nM PI3Kδ. Reactions were incubated for 210 min andterminated by the addition of 40 μL SPA beads suspended in quenchbuffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400μM ATP. The final concentration of SPA beads was 1.0 mg/mL. After theplate sealing, plates were shaken overnight at room temperature andcentrifuged at 1800 rpm for 10 minutes, the radioactivity of the productwas determined by scintillation counting on Topcount (Perkin-Elmer).IC₅₀ determination was performed by fitting the curve of percent controlactivity versus the log of the inhibitor concentration using theGraphPad Prism 3.0 software. The compound of Formula I was found have anIC₅₀ of ≤10 nM in the assay of Example A3.

Example B1: B Cell Proliferation Assay

To acquire B cells, human PBMC are isolated from the peripheral blood ofnormal, drug free donors by standard density gradient centrifugation onFicoll-Hypague (GE Healthcare, Piscataway, N.J.) and incubated withanti-CD19 microbeads (Miltenyi Biotech, Auburn, Calif.). The B cells arethen purified by positive immunosorting using an autoMacs (MiltenyiBiotech) according to the manufacture's instruction.

The purified B cells (2×10⁵/well/200 μL) are cultured in 96-wellultra-low binding plates (Corning, Corning, N.Y.) in RPMI1640, 10% FBSand goat F(ab′)2 anti-human IgM (10 μg/ml) (Invitrogen, Carlsbad,Calif.) in the presence of different amount of test compounds for threedays. [³H]-thymidine (1 μCi/well) (PerkinElmer, Boston, Mass.) in PBS isthen added to the B cell cultures for an additional 12 hours before theincorporated radioactivity is separated by filtration with water throughGF/B filters (Packard Bioscience, Meriden, Conn.) and measured by liquidscintillation counting with a TopCount (Packard Bioscience).

Example B2: Pfeiffer Cell Proliferation Assay

Pfeiffer cell line (diffuse large B cell lymphoma) are purchased fromATCC (Manassas, Va.) and maintained in the culture medium recommended(RPMI and 10% FBS). To measure the anti-proliferation activity of thecompounds, the Pfeiffer cells are plated with the culture medium (2×10³cells/well/per 200 μl) into 96-well ultra-low binding plates (Corning,Corning, N.Y.), in the presence or absence of a concentration range oftest compounds. After 3-4 days, [³H]-thymidine (1 μCi/well)(PerkinElmer, Boston, Mass.) in PBS is then added to the cell culturefor an additional 12 hours before the incorporated radioactivity isseparated by filtration with water through GF/B filters (PackardBioscience, Meridenj, Conn.) and measured by liquid scintillationcounting with a TopCount (Packard Bioscience).

Example B3: SUDHL-6 Cell Proliferation Assay

SUDHL-6 cell line (diffuse large B cell lymphoma) was purchased fromATCC (Manassas, Va.) and maintained in the culture medium recommended(RPMI and 10% FBS). To measure the anti-proliferation activity of thecompounds through ATP quantitation, the SUDHL-6 cells was plated withthe culture medium (5000 cells/well/per 200 μl) into 96-well polystyreneclear black tissue culture plate (Greiner-bio-one through VWR, NJ) inthe presence or absence of a concentration range of test compounds.After 3 days, Cell Titer-GLO Luminescent (Promega, Madison, Wis.) cellculture agent was added to each well for 10 minutes at room temperatureto stabilize the luminescent signal. This determines the number ofviable cells in culture based on quantitation of the ATP present, whichsignals the presence of metabolically active cells. Luminescence wasmeasured with the TopCount 384 (Packard Bioscience through Perkin Elmer,Boston, Mass.).

Example C: Akt Phosphorylation Assay

Ramos cells (B lymphocyte from Burkitts lymphoma) are obtained from ATCC(Manassas, Va.) and maintained in RPMI1640 and 10% FBS. The cells (3×10⁷cells/tube/3 mL in RPMI) are incubated with different amounts of testcompounds for 2 hrs at 37° C. and then stimulated with goat F(ab′)2anti-human IgM (5 μg/mL) (Invitrogen) for 17 minutes in a 37° C. waterbath. The stimulated cells are spun down at 4° C. with centrifugationand whole cell extracts are prepared using 300 L lysis buffer (CellSignaling Technology, Danvers, Mass.). The resulting lysates aresonicated and supernatants are collected. The phosphorylation level ofAkt in the supernatants are analyzed by using PathScan phospho-Akt1(Ser473) sandwich ELISA kits (Cell Signaling Technology) according tothe manufacturer's instruction.

Various modifications of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims. Each reference, including all patent,patent applications, and publications, cited in the present applicationis incorporated herein by reference in its entirety.

1-14. (canceled)
 15. A method of inhibiting an activity of a PI3Kkinase, comprising contacting the kinase with a salt which iscrystalline(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloric acid salt.
 16. The method of claim 15, wherein the PI3K isPI3Kδ.
 17. The method of claim 16, wherein said salt is a selectiveinhibitor for PI3Kδ over one or more of PI3Kα, PI3Kβ, or PI3Kγ.
 18. Amethod of treating a disease in a patient, wherein said disease isassociated with abnormal expression or activity of a PI3K kinase,comprising administering to said patient a therapeutically effectiveamount of crystalline(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloric acid salt.
 19. The method of claim 18, wherein the diseaseis selected from idiopathic thrombocytopenic purpura, autoimmunehemolytic anemia, vasculitis, systemic lupus erythematosus, lupusnephritis, pemphigus, autoimmune hemolytic anemia, membranousnephropathy, chronic lymphocytic leukemia, Non-Hodgkin lymphoma, hairycell leukemia, Mantle cell lymphoma, Burkitt lymphoma, small lymphocyticlymphoma, follicular lymphoma, lymphoplasmacytic lymphoma, extranodalmarginal zone lymphoma, Hodgkin's lymphoma, Waldenstrom'smacroglobulinemia, prolymphocytic leukemia, acute lymphoblasticleukemia, myelofibrosis, mucosa-associated lymphatic tissue lymphoma,B-cell lymphoma, mediastinal large B-cell lymphoma, lymphomatoidgranulomatosis, splenic marginal zone lymphoma, primary effusionlymphoma, intravascular large B-cell lymphoma, plasma cell leukemia,extramedullary plasmacytoma, smouldering myeloma, monoclonal gammopathyof undetermined significance and B cell lymphoma.
 20. The method ofclaim 19, wherein the method is a method of treating idiopathicthrombocytopenic purpura selected from relapsed idiopathicthrombocytopenic purpura and refractory idiopathic thrombocytopenicpurpura.
 21. The method of claim 19, wherein the method is a method oftreating vasculitis selected from Behçet's disease, Cogan's syndrome,giant cell arteritis, polymyalgia rheumatica, Takayasu's arteritis,Buerger's disease, central nervous system vasculitis, Kawasaki disease,polyarteritis nodosa, Churg-Strauss syndrome, mixed cryoglobulinemiavasculitis, essential mixed cryoglobulinemia vasculitis hepatitis Cvirus induced mixed cryoglobulinemia vasculitis, Henoch-Schönleinpurpura, hypersensitivity vasculitis, microscopic polyangiitis,Wegener's granulomatosis, and anti-neutrophil cytoplasm antibodyassociated systemic vasculitis.
 22. The method of claim 19, wherein themethod is a method of treating non-Hodgkin lymphoma selected fromrelapsed non-Hodgkin lymphoma, refractory non-Hodgkin lymphoma, andrecurrent follicular non-Hodgkin lymphoma.
 23. The method of claim 19,wherein the method is a method of treating B cell lymphoma, wherein saidB cell lymphoma is diffuse large B-cell lymphoma.
 24. The method ofclaim 19, wherein the method is a method of treating B cell lymphoma,wherein said B cell lymphoma is activated B-cell like diffuse large Bcell lymphoma, or germinal center B cell diffuse large B cell lymphoma.25. The method of claim 18, wherein said disease is osteoarthritis,restenosis, atherosclerosis, bone disorders, arthritis, diabeticretinopathy, psoriasis, benign prostatic hypertrophy, inflammation,angiogenesis, pancreatitis, kidney disease, inflammatory bowel disease,myasthenia gravis, multiple sclerosis, or Sjögren's syndrome.
 26. Themethod of claim 18, wherein said disease is rheumatoid arthritis,allergy, asthma, glomerulonephritis, lupus, or inflammation related toany of the aforementioned.
 27. The method of claim 26, wherein lupus issystemic lupus erythematosus or lupus nephritis.
 28. The method of claim18, wherein said disease is breast cancer, prostate cancer, coloncancer, endometrial cancer, brain cancer, bladder cancer, skin cancer,cancer of the uterus, cancer of the ovary, lung cancer, pancreaticcancer, renal cancer, gastric cancer, or a hematological cancer.
 29. Themethod of claim 28, wherein said hematological cancer is acutemyeloblastic leukemia or chronic myeloid leukemia.
 30. The method ofclaim 18, wherein said disease is acute lung injury or adult respiratorydistress syndrome. 31-85. (canceled)
 86. The method of claim 15, whereinthe crystalline(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloric acid salt has at least four XRPD peaks, in terms of2-theta, selected from about 11.3°, about 16.4°, about 21.0°, about23.0°, about 28.10, about 31.2°, and about 32.8°.
 87. The method ofclaim 18, wherein the crystalline(R)-4-(3-((S)-1-(4-amino-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-1-yl)ethyl)-5-chloro-2-ethoxy-6-fluorophenyl)pyrrolidin-2-onehydrochloric acid salt has at least four XRPD peaks, in terms of2-theta, selected from about 11.3°, about 16.4°, about 21.0°, about23.0°, about 28.10, about 31.2°, and about 32.8°.
 88. The method ofclaim 18, wherein the method is a method of treating chronic lymphocyticleukemia.
 89. The method of claim 18, wherein the method is a method oftreating hairy cell leukemia.
 90. The method of claim 18, wherein themethod is a method of treating Mantle cell lymphoma.
 91. The method ofclaim 18, wherein the method is a method of treating small lymphocyticlymphoma.
 92. The method of claim 18, wherein the method is a method oftreating follicular lymphoma.
 93. The method of claim 18, wherein themethod is a method of treating lymphoplasmacytic lymphoma.
 94. Themethod of claim 18, wherein the method is a method of treatingextranodal marginal zone lymphoma.
 95. The method of claim 18, whereinthe method is a method of treating myelofibrosis.
 96. The method ofclaim 18, wherein the method is a method of treating diffuse large Bcell lymphoma.
 97. The method of claim 18, wherein the method is amethod of treating activated B-cell like diffuse large B cell lymphoma.98. The method of claim 18, wherein the method is a method of treatinggerminal center B cell diffuse large B cell lymphoma.
 99. The method ofclaim 18, wherein the method is a method of treating Non-Hodgkinlymphoma.
 100. The method of claim 18, wherein the method is a method oftreating Hodgkin's lymphoma.
 101. The method of claim 18, wherein themethod is a method of treating lupus nephritis.
 102. The method of claim18, wherein the method is a method of treating systemic lupuserythematosus.
 103. The method of claim 18, wherein the method is amethod of treating pemphigus.
 104. The method of claim 18, wherein themethod is a method of treating autoimmune hemolytic anemia.
 105. Themethod of claim 18, wherein the method is a method of treating Sjögren'ssyndrome.